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Eur J Biochem, 1997 May 1, 245(3), 564 - 72
Identification of an RNA-binding-loop in the N-terminal region of signal-recognition-particle protein SRP19; Black SD et al.; Protein SRP19 is a 144-amino-acid polypeptide that associates intimately with the signal-recognition particle RNA (SRP RNA) and serves as an important structural and functional component of the SRP . We investigated the structure and RNA-binding activity of the human SRP19 protein by the use of comparative sequence analysis, high-stringency structure prediction, proteolytic susceptibility, and site-directed mutagenesis . SRP19 was found to consist of two distinct regions (called N-terminal and C-terminal regions) that are separated by a boundary of approximately 12-15 amino acid residues . Both regions contain an alpha-helix and several beta-strands that are connected by loops or turns . In agreement with the hypothetical model, proteolytic susceptibility demonstrated the predominant accessibility of two sites: one in a surface loop of the N-terminal region (YLNNKKTIAEGR33), and another site in the C-terminal tail at residues L129 and E133 . The RNA-binding activities of mutant polypeptides with changes of conserved lysines and arginines (mutants K27Q, R33Q and R34Q) demonstrated that the proteolytically accessible loop of the N-terminal region is in direct contact with the SRP RNA . In contrast, alteration of a certain basic amino acid residues in the C-terminal region (R83, K116 and R118), as well as a deletion of four amino acid residues located at the boundary between the two regions, had no effect on the RNA-binding ability . The structural model that emerges from our data is thematically similar to that of ribosomal protein S5, the N-domain of which contains a loop motif believed to interact with double-stranded RNA . The presence of a similar structural feature in protein SRP19 has significant implications for the structure and function of the SRP19-RNA complex.

Neuron, 1997 May, 18(5), 711 - 22
The vibrator mutation causes neurodegeneration via reduced expression of PITP alpha: positional complementation cloning and extragenic suppression; Hamilton BA et al.; The mouse vibrator mutation causes an early-onset progressive action tremor, degeneration of brain stem and spinal cord neurons, and juvenile death . We cloned the vibrator mutation using an in vivo positional complementation approach and complete resequencing of the resulting 76 kb critical region from vibrator and its parental chromosome . The mutation is an intracisternal A particle retroposon insertion in intron 4 of the phosphatidylinositol transfer protein alpha gene, causing a 5-fold reduction in RNA and protein levels . Expression of neurofilament light chain is also reduced in vibrator, suggesting one signaling pathway that may underlie vibrator pathology . The vibrator phenotype is suppressed in one intercross . We performed a complete genome scan and mapped a major suppressor locus (Mvb-1) to proximal chromosome 19.

Bioessays, 1997 May, 19(5), 367 - 70
Telomeres, not the end of the story; Gotta M et al.; Transcription in organisms as diverse as yeast and mammals is subject to chromosomal position effects that result in heritable and variegated patterns of gene expression . Two recent studies have employed a reversible protein-DNA crosslinking method to identify the structural components of heterochromatin in budding yeast . The results show that a complex containing the proteins Rap1, Sir2p, Sir3p and Sir4p is physically associated with nucleosomes at telomere proximal regions, but that the repressive chromatin structure extended by Sir3p overexpression has a different composition.

DNA Cell Biol, 1997 May, 16(5), 663 - 9
Isolation of a cDNA encoding a Kex2-like endoprotease with homology to furin from the nematode Caenorhabditis elegans; Gomez-Saladin E et al.; A cDNA was isolated from the nematode Caenorhabditis elegans that encodes an endoprotease which is a member of the Kex2 family of serine endoproteases . Degenerate oligonucleotide primers were designed based on conserved regions within the active sites of known Kex2-like endoproteases, and were used for reverse transcription-polymerase chain reaction (RT-PCR) of poly(A)+RNA isolated from C . elegans . A PCR product was isolated that had homology to the active sites of known furin endoproteases, and was used as a probe to screen a C . elegans cDNA library . A Kex2-like endoprotease (CelfurPC) which encoded a 692-amino-acid pre-proendoprotease, was identified . The deduced amino acid sequence for the catalytic domain of CelfurPC is homologous to the known Kex2-like endoproteases, with strongest structural homology to the furin/PACE4 family . However, all furins and PACE4 proteins contain a characteristic cysteine-rich domain, and all furins contain a transmembrane domain, neither of which is present in the CelfurPC protein . CelfurPC may thus represent a new class of Kex2-like endoprotease.

EMBO J, 1997 May 1, 16(9), 2493 - 506
Histone octamer function in vivo: mutations in the dimer-tetramer interfaces disrupt both gene activation and repression; Santisteban MS et al.; Within the core histone octamer each histone H4 interacts with each H2A-H2B dimer subunit through two binding surfaces . Tyrosines play a central role in these interactions with H4 tyrosines 72 and 88 contacting one H2A-H2B dimer subunit, and tyrosine 98 contacting the other . To investigate the roles of these interactions in vivo, we made site-directed amino acid substitutions at each of these tyrosine residues . Elimination of either set of interactions is lethal, suggesting that binding of the tetramer to both dimers is essential . Temperature-sensitive mutants were obtained through single amino acid substitutions at each of the tyrosines . The mutants show both strong positive and negative effects on transcription . Positive effects include Spt- and Sin-phenotypes resulting from mutations at each of the three tyrosines . One allele has a strong negative effect on the expression of genes essential for the G1 cell cycle transition . At restrictive temperature, mutant cells fail to express the CLN1, CLN2, SWI4 and SWI6 genes, and have reduced levels of CLN3 mRNA . These results demonstrate the critical role of histone dimer-tetramer interactions in vivo, and define their essential role in the expression of genes regulating G1 cell cycle progression.

EMBO J, 1997 May 1, 16(9), 2251 - 61
The linker region of the ABC-transporter Ste6 mediates ubiquitination and fast turnover of the protein; Kolling R et al.; Upon block of endocytosis, the a-factor transporter Ste6 accumulates in a ubiquitinated form at the plasma membrane . Here we show that the linker region, which connects the two homologous halves of Ste6, contains a signal which mediates ubiquitination and fast turnover of Ste6 . This signal was also functional in the context of another plasma membrane protein . Deletion of an acidic stretch in the linker region ('A-box') strongly stabilized Ste6 . The A-box contains a sequence motif ('DAKTI') which resembles the putative endocytosis signal of the alpha-factor receptor Ste2 ('DAKSS') . Deletion of the DAKTI sequence also stabilized Ste6 but, however, not as strongly as the A-box deletion . There was a correlation between the half-life of the mutants and the degree of ubiquitination: while ubiquitination of the deltaDAKTI mutant was reduced compared with wild-type Ste6, no ubiquitination could be detected for the more stable deltaA-box variant . Loss of ubiquitination seemed to affect Ste6 trafficking . In contrast to wild-type Ste6, which was associated mainly with internal membranes, the ubiquitination-deficient mutants accumulated at the plasma membrane, as demonstrated by immunofluorescence and cell fractionation experiments . These findings suggest that ubiquitination is required for efficient endocytosis of Ste6 from the plasma membrane.

EMBO J, 1997 May 1, 16(9), 2227 - 39
A novel structural model for regulation of clathrin function; Pishvaee B et al.; The distinctive triskelion shape of clathrin allows assembly into polyhedral lattices during the process of clathrin-coated vesicle formation . We have used random and site-directed mutagenesis of the yeast clathrin heavy chain gene (CHC1) to characterize regions which determine Chc trimerization and binding to the clathrin light chain (Clc) subunit . Analysis of the mutants indicates that mutations in the trimerization domain at the triskelion vertex, as well as mutations in the adjacent leg domain, frequently influence Clc binding . Strikingly, one mutation in the trimerization domain enhances the association of Clc with Chc . Additional mutations in the trimerization domain, in combination with mutations in the adjacent leg domain, exhibit severe defects in Clc binding while maintaining near normal trimerization properties . The position of these trimerization domain mutations on one face of a putative alpha-helix defines a region on the trimer surface that interacts directly with Clc . These results suggest that Clc extends into the Chc trimerization domain from the adjacent leg, thereby bridging the two domains . On the basis of this conclusion, we propose a new model for the organization of the triskelion vertex which provides a structural basis for regulatory effects of Clc on clathrin function.

EMBO J, 1997 May 1, 16(9), 2205 - 16
Multiple interactions of components mediating preprotein translocation across the inner mitochondrial membrane; Bomer U et al.; The protein transport machinery of the inner mitochondrial membrane contains three essential Tim proteins . Tim17 and Tim23 are thought to build a preprotein translocation channel, while Tim44 transiently interacts with the matrix heat shock protein Hsp70 to form an ATP-driven import motor . For this report we characterized the biogenesis and interactions of Tim proteins . (i) Import of the precursor of Tim44 into the inner membrane requires mtHsp70, whereas import and inner membrane integration of the precursors of Tim17 and Tim23 are independent of functional mtHsp70 . (ii) Tim17 efficiently associates with Tim23 and mtHsp70, but only weakly with Tim44 . (iii) Depletion of Tim44 does not affect the co-precipitation of Tim17 with antibodies directed against mtHsp70 . (iv) Tim23 associates with both Tim44 and Tim17, suggesting the presence of two Tim23 pools in the inner membrane, a Tim44-Tim23-containing sub-complex and a Tim23-Tim17-containing sub-complex . (v) The association of mtHsp70 with the Tim23-Tim17 sub-complex is ATP sensitive and can be distinguished from the mtHsp70-Tim44 interaction by the differential influence of an amino acid substitution in mtHsp70 . (vi) Genetic evidence, suppression of the protein import defect of a tim17 yeast mutant by overexpression of mtHsp70 and synthetic lethality of conditional mutants in the genes of Tim17 and mtHsp70, supports a functional interaction of mtHsp70 with Tim17 . We conclude that the protein transport machinery of the mitochondrial inner membrane consists of dynamically interacting sub-complexes, each of which transiently binds mtHsp70.

J Nat Prod, 1997 May, 60(5), 507 - 10
Two new nitrogenous sesquiterpenes from the sponge Axinyssa aplysinoides; Patil AD et al.; Bioassay-guided fractionation of the EtOAc extract of the Palauan sponge Axinyssa aplysinoides yielded two novel alkaloids, 1 and 2 . The structure of 2-(formylamino)trachyopsane (1) was determined by X-ray analysis; and the structure of N-phenethyl-N'-2-trachyopsanylurea (2), by interpretation of the spectral data.

J Nat Prod, 1997 May, 60(5), 478 - 81
A bioactive seco-rosane diterpenoid from Vellozia candida; Valente LM et al.; Bioassay-directed fractionation of the bioactive alcoholic extracts of Vellozia candida yielded a new 6,7-seco-rosane diterpenoid, candidalactone (1), which showed moderate toxicity toward DNA repair-deficient mutants of Saccharomyces cerevisiae . Another new but inactive rosane diterpenoid, candidenodiol (3), was also obtained.

Plant Cell, 1997 May, 9(5), 759 - 71
The Arabidopsis ABSCISIC ACID-INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction; Leung J et al.; Abscisic acid (ABA) mediates seed maturation and adaptive responses to environmental stress . In Arabidopsis, the ABA-INSENSITIVE1 (ABI1) protein phosphatase 2C is required for proper ABA responsiveness both in seeds and in vegetative tissues . To determine whether the lack of recessive alleles at the corresponding locus could be explained by the existence of redundant genes, we initiated a search for ABI1 homologs . One such homolog turned out to be the ABI2 locus, whose abi2-1 mutation was previously known to decrease ABA sensitivity . Whereas abi1-1 is (semi)dominant, abi2-1 has been described as recessive and maternally controlled at the germination stage . Unexpectedly, the sequence of the abi2-1 mutation showed that it converts Gly-168 to Asp, which is precisely the same amino acid substitution found in abi1-1 and at the coincidental position within the ABI1 phosphatase domain (Gly-180 to Asp) . In vitro assays and functional complementation studies in yeast confirmed that the ABI2 protein is an active protein phosphatase 2C and that the abi2-1 mutation reduced phosphatase activity as well as affinity to Mg2+ . Although a number of differences between the two mutants in adaptive responses to stress have been reported, quantitative comparisons of other major phenotypes showed that the effects of both abi1-1 and abi2-1 on these processes are nearly indistinguishable . Thus, the homologous ABI1 and ABI2 phosphatases appear to assume partially redundant functions in ABA signaling, which may provide a mechanism to maintain informational homeostasis.

Mutat Res, 1997 May 1, 383(3), 197 - 203
XPC interacts with both HHR23B and HHR23A in vivo; Li L et al.; XP group C protein (XPC) and a human homologue of RAD23, HHR23B, have previously been shown to copurify in a tightly associated complex . Here, we show that XPC interacts in vivo, by means of the yeast two-hybrid system, with both HHR23B and a second homologue of RAD23, HHR23A . Domain mapping studies have revealed that both RAD23 homologues interact with XPC at the same highly conserved region in the C-terminal half of the protein . XPC mutants deleted within this domain and that are highly deficient in binding both RAD23 homologues are also highly defective in complementing XPC cells in vivo . Domain mapping studies have also identified a region in the N-terminal half of HHR23B that contains the XPC interactive site . This domain is highly conserved among HHR23B, HHR23A, and RAD23.

Curr Genet, 1997 May, 31(5), 401 - 7
A mutation in cytochrome oxidase subunit 2 restores respiration of the mutant pet ts1402; Meyer W et al.; The yeast PET1402/OXA1 gene encoding a 44.8-kDa protein is required for mitochondrial biogenesis . Substitution of Leu240 to serine in the protein results in an accumulation of the precursor form of the mitochondrially encoded subunit 2 of cytochrome oxidase (Cox2) and temperature-sensitive respiration . This temperature sensitivity can be suppressed by a mutation in the cox2 gene changing Ala189 of the Cox2 protein to proline . In the cox2-ts1402 double mutant respiration is restored without removal of the Cox2 pre-sequence . The suppression suggests an interaction of the Pet1402 protein with the cytochrome oxidase complex . Antibodies raised against the predicted C-terminus and the tagged N-terminus of the Pet1402 protein reacted with a 37-kDa polypeptide . This protein, present in the mitochondrial fraction, is localized within the inner membrane . The difference in size can be explained by the removal of the predicted mitochondrial-targeting sequence from the Pet1402 protein . The mitochondrial localization of the protein points to a direct interaction with the cytochrome oxidase complex.

Oncogene, 1997 May 1, 14(17), 2091 - 8
Cross-family interaction between the bHLHZip USF and bZip Fra1 proteins results in down-regulation of AP1 activity; Pognonec P et al.; Heterodimerization among the basic-leucine zipper (bZIP) proteins or among the basic-helix-loop-helix-leucine zipper (bHLHZip) proteins confers a multitude of combinational activities to these transcription factors . To further examine the function of the bHLHZip protein, USF, we screened for cellular proteins which could directly interact with USF using the yeast two-hybrid system . A bZip protein, Fra1, was found to efficiently interact with USF . USF specifically interacts with Fra1 but not with other closely related family members, c-Fos, Fra2, FosB, or with c-Jun . Both the bHLHZip and the N-terminal regions of Fra1 are required for efficient interaction with USF . In vivo association between USF and Fra1 has been demonstrated by co-immunoprecipitation . Expression of exogenous USF led to a decrease in AP1-dependent transcription in F9 cells . Co-expression of exogenous Fra1 restored the AP1 activity in a dose-dependent manner . These data show that USF and Fra1 physically and functionally interact demonstrating that cross-talk occurs between factors of distantly related transcription families.

Oncogene, 1997 May 1, 14(17), 2047 - 57
YSK1, a novel mammalian protein kinase structurally related to Ste20 and SPS1, but is not involved in the known MAPK pathways; Osada S et al.; To clarify the upstream regulatory mechanism of mitogen-activated protein kinase (MAPK), we performed the reverse transcriptase-based polymerase chain reaction (RT-PCR) with degenerate primers synthesized based on sequences conserved among the kinase domains of yeast MAPK kinase kinases (MAPKKKs), Stell, Bck1, and Byr2 . We isolated several mammalian cDNA fragments that encode kinase subdomains sharing significant sequence homology with yeast MAPKKKs . Subsequent screening of a HeLa cell cDNA library using one of these cDNA fragments as a probe resulted in the isolation of a full-length cDNA that encodes a novel protein kinase . The catalytic domain sequence of this gene product is closely related to those of budding yeast Sps1 and Ste20 protein kinases . Thus, we call this protein YSK1 (Yeast Sps1/Ste20-related Kinase 1) . The transcript of YSK1 was detected in a wide range of tissues and cells . Immunoprecipitated YSK1 shows protein kinase activity . Although YSK1 is significantly similar in its kinase domain to kinases of the yeast and mammalian MAPK pathways, the overexpression of YSK1 did not lead to the activation of the ERK (extracellular signal-regulated kinase) pathway, JNK (c-Jun NH2-terminal kinase)/SAPK (stress-activated protein kinase) pathway, or p38/Mpk2 pathway . These results suggest that YSK1 may be involved in the regulation of a novel intracellular signaling pathway.

Plant Physiol, 1997 May, 114(1), 315 - 24
Characterization of AtSEC12 and AtSAR1 . Proteins likely involved in endoplasmic reticulum and Golgi transport; Bar-Peled M et al.; Transport of cargo proteins from the endoplasmic reticulum (ER) to the cis-Golgi network is mediated by protein-coated vesicles . The coat, called COPII coat, consists of proteins that are recruited from the cytosol and interact with integral membrane proteins of the ER . In yeast, both cytosolic proteins (Sec13/31, Sec23/24, and Sar1) and ER-associated proteins (Sec12 and others) have been purified and characterized and it has been possible to demonstrate transport vesicle formation in vitro . Arabidopsis thaliana homologs of Sar1 and Sec12 have recently been identified, but little is known about the properties of the proteins or their subcellular distribution . Here we demonstrate that AtSAR1, a 22-kD protein that binds GTP, and AtSEC12, a 43-kD GTP-exchange protein, are both associated with the ER . However, about one-half of the cellular AtSAR1 is present in the cytosol . When AtSAR1 is overexpressed in transgenic plants, the additional protein is also cytosolic . When tissue-culture cells are cold-shocked (12 h at 8 degrees C), AtSAR1 levels appeared to decline and a larger proportion of the total protein was found in the cytosol . Given the known function of AtSAR1 in yeast, we propose that the amount of ER-associated AtSAR1 is an indication of the intensity of the secretory process . Thus, we expect that such a cold shock will adversely affect ER-to-Golgi transport of proteins.

Arterioscler Thromb Vasc Biol, 1997 May, 17(5), 996 - 1002
A novel mosaic protein containing LDL receptor elements is highly conserved in humans and chickens; Morwald S et al.; Certain receptors belonging to the LDL receptor (LDLR) gene family appear to constitute a newly identified branch whose members are expressed in brain, in addition to other tissues . In support of this concept, we have now discovered the expression and delineated the molecular structures of a representative of this emerging branch from two such diverse species as human and chicken . This membrane receptor, called LR11 and thus far only known to exist in the rabbit, is a complex seven-domain mosaic protein containing, among other structural elements, a cluster of 11 LDLR ligand-binding repeats and a domain with homology to VPS10, a yeast receptor for vacuolar protein sorting . Cytoplasmic signature sequences define the receptor as competent for endocytosis . The most striking properties of LR11s are their (1) high degree of structural conservation (>80% identity among mammals and birds), with 100% identity in the membrane-spanning and cytoplasmic domains of rabbit and human; (2) lack of regulation by cholesterol and estrogen; and (3) expression in brain . The features of LR11 suggest important roles in intercellular and intracellular ligand transport processes, some of which it may share with other brain-specific LDLR family members.

J Gen Virol, 1997 May, 78 ( Pt 5), 1083 - 6
Hepatitis B virus preS1 functions as a transcriptional activation domain; Kim HS et al.; Hepatitis B virus (HBV) preS1 fused to the GAL4 DNA-binding domain functioned as a transcriptional activation domain in yeast and mammalian cells . The GAL4-preS1 fusion proteins derived from the preS1 of all three tested HBV subtypes (adr, adw and ayw) specifically activated the transcription of a lacZ or chloramphenicol acetyltransferase reporter gene linked to a GAL4-responsive promoter in transient transfection assays using yeast or HepG2 cells, respectively . Deletion analyses showed that the segments of preS1 from residues 21 to 90 and from residues 21 to 56 are sufficient and essential for the activity, respectively . Stable expression of GAL4-preS1 in Chinese hamster ovary cells also produced transactivator activity . These results suggest that preS1 fused to any DNA-binding domain of transcription factors would have transactivation potential.

RNA, 1997 May, 3(5), 489 - 97
Importance of structural features for tRNA(Met) identity; Aphasizhev R et al.; We showed previously that the tRNA tertiary structure makes an important contribution to the identity of yeast tRNA(Met) (Senger B, Aphasizhev R, Walter P, Fasiolo F, 1995, J Mol Biol 249:45-58) . To learn more about the role played by the tRNA framework, we analyzed the effect of some phosphodiester cleavages and 2'OH groups in tRNA binding and aminoacylation . The tRNA is inactivated provided the break occurs in the central core region responsible for the tertiary fold or in the anticodon stem/loop region . We also show that, for tRNA(Met) to bind, the anticodon loop, but not the anticodon stem, requires a ribosephosphate backbone . A tertiary mutant of yeast tRNA(Met) involving interactions from the D- and T-loop unique to the initiator species fails to be aminoacylated, but still binds to yeast methionyl-tRNA synthetase . In the presence of 10 mM MgCl2, the mutant transcript has a 3D fold significantly stabilized by about 30 degrees C over a wild-type transcript as deduced from the measure of their T(m) values . The k(cat) defect of the tRNA(Met) mutant may arise from a failure to overcome an increase of the free energetic cost of distorting the more stable tRNA structure and/or a tRNA based MetRS conformational change required for formation of transition state of aminoacylation.

Brain Res Mol Brain Res, 1997 May, 45(2), 349 - 52
Localization of gene expression for phosphatidylinositol transfer protein in the brain of developing and mature rats; Utsunomiya A et al.; Gene expression for alpha- and beta-isoforms of phosphatidylinositol transfer protein (PITP) was examined using in situ hybridization histochemistry in developing and mature rat brains . During embryonic and early post-natal stages, gene expression for both PITP-alpha and -beta were detected widely throughout the entire neuraxis . In the adult brain, the expression for PITP-alpha was positive in almost all neurons throughout the entire brain while the expression for PITP-beta markedly decreased in the entire gray matter regions except for the cerebellar cortex . By comparison with the previous findings on the expression for various molecules involved in the PI turnover, the present finding suggests that PITP is involved more intimately in some differentiation-related functions of immature neurons than those of mature neurons in co-operation with PI-related molecules and that PITPs exert their functions in adult brain in concert with PLCs in subtype-preferable inter-relation.

Protein Sci, 1997 May, 6(5), 956 - 70
Homology modeling using simulated annealing of restrained molecular dynamics and conformational search calculations with CONGEN: application in predicting the three-dimensional structure of murine homeodomain Msx-1; Li H et al.; We have developed an automatic approach for homology modeling using restrained molecular dynamics and simulated annealing procedures, together with conformational search algorithms available in the molecular mechanics program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137-168) . The accuracy of the method is validated by "predicting" structures of two homeodomain proteins with known three-dimensional structures, and then applied to predict the three-dimensional structure of the homeodomain of the murine Msx-1 transcription factor . Regions of the unknown protein structure that are highly homologous to the known template structure are constrained by "homology distance constraints," whereas the conformations of nonhomologous regions of the unknown protein are defined only by the potential energy function . A full energy function (excluding explicit solvent) is employed to ensure that the calculated structures have good conformational energies and are physically reasonable . As in NMR structure determinations, information on the consistency of the structure prediction is obtained by superposition of the resulting family of protein structures . In this paper, our homology modeling algorithm is described and compared with related homology modeling methods using spatial constraints derived from the structures of homologous proteins . The software is then used to predict the DNA-bound structures of three homeodomain proteins from the X-ray crystal structure of the engrailed homeodomain protein (Kissinger CR et al., 1990, Cell 63:579-590) . The resulting backbone and side-chain conformations of the modeled yeast Mat alpha 2 and D . melanogaster Antennapedia homeodomains are excellent matches to the corresponding published X-ray crystal (Wolberger C et al., 1991, Cell 67:517-528) and NMR (Billeter M et al., 1993, J Mol Biol 234:1084-1097) structures, respectively . Examination of these structures of Msx-1 reveals a network of highly conserved surface salt bridges that are proposed to play a role in regulating protein-protein interactions of homeodomains in transcription complexes.

Arch Biochem Biophys, 1997 May 1, 341(1), 112 - 21
Fatty acid binding protein: stimulation of microsomal phosphatidic acid formation; Jolly CA et al.; The effect of fatty acid binding proteins (FABPs) on two key steps of microsomal phosphatidic acid formation was examined . Rat liver microsomes were purified by size-exclusion chromatography to remove endogenous cytosolic fatty acid and fatty acyl-CoA binding proteins while recombinant FABPs were used to avoid cross-contamination with such proteins from native tissue . Neither rat liver (L-FABP) nor rat intestinal fatty acid binding protein (I-FABP) stimulated liver microsomal fatty acyl-CoA synthase . In contrast, L-FABP and I-FABP enhanced microsomal conversion of {14C}oleoyl-CoA and glycerol 3-phosphate to {14C}phosphatidic acid by 18- and 7-fold, respectively . The mechanism for this stimulation, especially by I-FABP, is not known . However, several observations presented here suggest that, like L-FABP, I-FABP may interact with fatty acyl-CoA and thereby stimulate enzyme activity . First, I-FABP decreased microsomal membrane-bound oleoyl-CoA . Second, oleoyl-CoA displaced I-FABP bound fluorescent fatty acid, cis-parinaric acid, with Ki of 5.3 microM and 1.1 sites . Third, oleoyl-CoA decreased I-FABP tryptophan fluorescence with a Kd of 4.2 microM . Fourth, oleoyl-CoA red shifted emission spectra of acrylodated I-FABP, a sensitive marker of I-FABP interactions with ligands . In summary, the results demonstrate for the first time that both L-FABP and I-FABP stimulate liver microsomal phosphatidic acid formation by enhancing synthesis of phosphatidate from fatty acyl-CoA and glycerol 3-phosphate.

Arch Biochem Biophys, 1997 May 1, 341(1), 104 - 11
Expression of CYP71B7, a cytochrome P450 expressed sequence Tag from Arabidopsis thaliana; Maughan JA et al.; The systematic sequencing of anonymous cDNA clones (expressed sequence tags or ESTs) from the plant Arabidopsis thaliana has identified a number of cDNAs with similarity to known cytochrome P450 sequences . The partial sequence of one of these cDNAs, 5G6, indicated that it was likely to encode a full-length cytochrome P450 monooxygenase (cyt P450) sequence . In this paper we describe the complete sequence of this clone, which has been designated CYP71B7 in accordance with the nomenclature for the cyt P450 gene superfamily . The cDNA was used to determine the pattern of expression of the corresponding gene in A . thaliana . Northern hybridization analysis indicated that maximal expression of CYP71B7 occurred in rosette leaves . Weaker hybridizing bands were also detected by Northern analysis of RNA from roots, leaves, flowers, and siliques . No expression could be detected in stem tissue . Southern analysis indicated that the CYP71B7 gene was likely to exist as a single copy in the genome of A . thaliana . CYP71B7 was expressed episomally in yeast, and microsomes prepared from transgenic yeast exhibited a carbon monoxide difference spectrum characteristic of cyt P450 . Microsomes from yeast expressing CYP71B7 were assayed for enzymatic activity with synthetic model cyt P450 substrates . Microsomes from yeast cells expressing CYP71B7 or those from control cells exhibited no detectable NADPH-supported 7-ethoxycoumarin or 7-ethoxyresorufin deethylase activities . However, in the presence of cumene hydroperoxide, activity was observed with microsomes from cells expressing CYP71B7 with 7-ethoxycoumarin as substrate . Organic hydroperoxides are well known to support cyt P450 catalysis in the absence of electrons from NADPH . The yeast microsomes contained high levels of endogenous NADPH-ferricytochrome P450 reductase (CPR) activity . The data suggest that this A . thaliana cyt P450, although expressed in an active form, is incapable of accepting electrons from the endogenous yeast CPR protein.

Appl Environ Microbiol, 1997 May, 63(5), 1661 - 6
ord1, an oxidoreductase gene responsible for conversion of O-methylsterigmatocystin to aflatoxin in Aspergillus flavus; Prieto R et al.; Among the enzymatic steps in the aflatoxin biosynthetic pathway, the conversion of O-methylsterigmatocystin to aflatoxin has been proposed to be catalyzed by an oxidoreductase . Transformants of Aspergillus flavus 649WAF2 containing a 3.3-kb genomic DNA fragment and the aflatoxin biosynthesis regulatory gene aflR converted exogenously supplied O-methylsterigmatocystin to aflatoxin B1 . A gene, ord1, corresponding to a transcript of about 2 kb was identified within the 3.3-kb DNA fragment . The promoter region presented a putative AFLR binding site and a TATA sequence . The nucleotide sequence of the gene revealed an open reading frame encoding a protein of 528 amino acids with a deduced molecular mass of 60.2 kDa . The gene contained six introns and seven exons . Heterologous expression of the ord1 open reading frame under the transcriptional control of the Saccharomyces cerevisiae galactose-inducible gal1 promoter results in the ability to convert O-methylsterigmatocystin to aflatoxin B1 . The data indicate that ord1 is sufficient to accomplish the last step of the aflatoxin biosynthetic pathway . A search of various databases for similarity indicated that ord1 encodes a cytochrome P-450-type monooxygenase, and the gene has been assigned to a new P-450 gene family named CYP64.

J Clin Endocrinol Metab, 1997 May, 82(5), 1440 - 6
Steroid 21-hydroxylase autoantibodies: measurements with a new immunoprecipitation assay; Tanaka H et al.; Autoantibodies (Abs) to steroid 21-hydroxylase (21-OH) are a major component of adrenal cortex Abs and are characteristic of autoimmune Addison's disease . We have developed a new method for measuring Abs to 21-OH based on 125I-labeled recombinant human 21-OH produced in yeast . With this assay, 21-OH Abs were detected in 43 of 60 (72%) sera from patients with isolated Addison's disease, 11 of 12 (92%) autoimmune polyglandular syndrome type I sera, 27 of 27 (100%) autoimmune polyglandular syndrome type II sera, and 24 of 30 (80%) sera from patients who were positive for adrenal cortex antibodies by immunofluorescence but had no overt Addison's disease . 21-OH Abs were found by 125I assay in 4 of 150 (2.7%) sera from patients with insulin-dependent diabetes mellitus, 1 of 77 (1.3%) Graves' sera, 1 of 67 (1.5%) Hashimoto's sera, and 6 of 243 (2.5%) sera from healthy blood donors . 21-OH Abs were not detected in 9 sera from patients with Addison's disease due to tuberculosis, 32 sera from patients with noninsulin-dependent diabetes mellitus, 35 sera from patients with myasthenia gravis, or 17 sera from patients with premature ovarian failure . There was good agreement between the 125I-labeled 21-OH assay and an assay based on 35S-labeled 21-OH produced in an in vitro transcription/translation system (r = 0.86; n = 129; P < 0.001) . In the case of sera from patients with Addison's disease, insulin-dependent diabetes mellitus, Graves' disease, and Hashimoto's disease and from healthy blood donors that were low positive in the 125I assay, neutralization studies with unlabeled 21-OH confirmed the presence of specific 21-OH Abs . Overall, the 21-OH Ab assay based on 125I-labeled 21-OH showed good sensitivity, precision, and disease group specificity . This, combined with a simple assay protocol and the convenience of 125I handling and counting, make it attractive for routine use . Further investigations with the new assay should allow wider assessment of the prevalence and pattern of inheritance of adrenal autoimmunity . In addition, studies of the effect of treatment or possible preventative measures on 21-OH Ab levels in individuals without overt adrenal failure may suggest ways of delaying the onset of autoimmune Addison's disease.

Nat Genet, 1997 May, 16(1), 88 - 92
Mutations in PMM2, a phosphomannomutase gene on chromosome 16p13, in carbohydrate-deficient glycoprotein type I syndrome (Jaeken syndrome)
Matthijs G, Schollen E, Pardon E, Veiga-Da-Cunha M, Jaeken J, Cassiman JJ, Van Schaftingen E.
Carbohydrate-deficient glycoprotein syndrome type 1 (CDG1 or Jaeken syndrome) is the prototype of a class of genetic multisystem disorders characterized by defective glycosylation of glycoconjugates . It is mostly a severe disorder which presents neonatally . There is a severe encephalopathy with axial hypotonia, abnormal eye movements and pronounced psychomotor retardation, as well as a peripheral neuropathy, cerebellar hypoplasia and retinitis pigmentosa . The patients show a peculiar distribution of subcutaneous fat, nipple retraction and hypogonadism . There is a 20% lethality in the first years of life due to severe infections, liver insufficiency or cardiomyopathy . CDG1 shows an autosomal recessive mode of inheritance and has been mapped to chromosome 16p . Most patients show a deficiency of phosphomannomutase (PMM)8, an enzyme necessary for the synthesis of GDP-mannose . We have cloned the PMM1 gene, which is on chromosome 22q13 (ref.9) . We now report the identification of a second human PMM gene, PMM2, which is located on 16p13 and which encodes a protein with 66% identity to PMM1 . We found eleven different missense mutations in PMM2 in 16 CDG1 patients from different geographical origins and with a documented phosphomannomutase deficiency . Our results give conclusive support to the biochemical finding that the phosphomannomutase deficiency is the basis for CDG1.

Nature, 1997 May 1, 387(6628), 49 - 55
Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression; Alland L et al.; Normal mammalian growth and development are highly dependent on the regulation of the expression and activity of the Myc family of transcription factors . Mxi1-mediated inhibition of Myc activities requires interaction with mammalian Sin3A or Sin3B proteins, which have been purported to act as scaffolds for additional co-repressor factors . The identification of two such Sin3-associated factors, the nuclear receptor co-repressor (N-CoR) and histone deacetylase (HD1), provides a basis for Mxi1/Sin3-induced transcriptional repression and tumour suppression.

Nature, 1997 May 1, 387(6628), 43 - 8
A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression; Heinzel T et al.; Transcriptional repression by nuclear receptors has been correlated to binding of the putative co-repressor, N-CoR . A complex has been identified that contains N-CoR, the Mad presumptive co-repressor mSin3, and the histone deacetylase mRPD3, and which is required for both nuclear receptor- and Mad-dependent repression, but not for repression by transcription factors of the ets-domain family . These data predict that the ligand-induced switch of heterodimeric nuclear receptors from repressor to activator functions involves the exchange of complexes containing histone deacetylases with those that have histone acetylase activity.

Mol Endocrinol, 1997 May, 11(5), 587 - 94
Estrogen receptor residues required for stereospecific ligand recognition and activation; Bocchinfuso WP et al.; The mouse estrogen receptor (mER) has been shown to exhibit stereospecific binding of certain stilbene estrogen agonists . The region of the mER involved in the stereochemical recognition of ligands was further defined using a stilbene isomer, Indenestrol B (IB) . The IB compound has a chiral carbon bearing an ethyl substituent, and the wild type uterine mER has been shown to bind the enantiomers, IB-S and IB-R, with similar affinity . The wild type mER expressed in yeast exhibited a very minor preference for IB-S in transactivation (1.5-fold lower half-maximal dose than IB-R) . The IB enantiomers could then be used to determine whether stereochemically distinct compounds with similar transcriptional activity utilize different amino acids in AF-2 for transactivation . Mutant mERs with glycine substitutions at Met521, His528, Met532, and Val537 were expressed in yeast and measured for IB-S- and IB-R-induced transactivation and ligand binding . The M532G mER showed a 124-fold and 50-fold reduction in transactivation induced by IB-S and IB-R, respectively, without a corresponding change in their ligand-binding affinities . Therefore, Met532 is required for transactivation induced by both IB enantiomers but does not discriminate based on stereospecificity . In contrast, the H528G mER displayed a gross change in stereospecific ligand recognition as illustrated by a 110-fold reduction in transactivation by IB-S and only a 8.8-fold decrease in activity by IB-R . As a result, H528G mER displayed a switch in ligand preference such that IB-R was now 8-fold more active than IB-S in transactivation . Therefore, His528 appears largely involved in transactivation specifically induced by IB-S but has a minimal influence in IB-S ligand binding . The remaining mutant mERs displayed wild type ligand binding and transactivation properties for the IB enantiomers illustrating no stereospecific recognition . These results imply that individual IB enantiomers bind to the mER with similar affinity but utilize at least one different amino acid within the AF-2 domain for signal transduction . The binding of stereochemically distinct ligands may alter the tertiary structure of the mER and cause repositioning of the AF-2 region that mediates transcription of specific genes and/or affect the binding of receptor-associated proteins, such as coactivators, which could influence transcription.

Genetics, 1997 May, 146(1), 121 - 33
FAR1, a negative regulatory locus required for the repression of the nitrate reductase gene in Chlamydomonas reinhardtii; Zhang D et al.; In Chlamydomonas reinhardtii, the genes required for nitrate assimilation, including the gene encoding nitrate reductase (NIT1), are subject to repression by ammonia . To study the mechanism of ammonia repression, we employed two approaches to search for mutants with defective repression of NIT1 gene expression . (1) PF14, a gene required for flagellar function, was used as a reporter gene for expression from the NIT1 promoter . When introduced into a pf14 mutant host, the NIT1;PF14 chimeric construct produced a transformant (T10-10B) with a conditional swimming phenotype . Spontaneous mutants with defective ammonia repression of the NIT1 promoter were screened for by isolating cells that gained constitutive motility . (2) Insertional mutagenesis was performed, followed by screening for chlorate sensitivity in the presence of ammonia ion . One insertional mutant and six spontaneous mutants were allelic and defined a new gene, FAR1 (free from ammonia repression) . FAR1 was mapped to Linkage Group I, 7.7 cM to the right of the centromere . The far1-1 mutant strain was used to clone DNA adjacent to the site of plasmid insertion, which was then used as a hybridization probe to clone the FAR1 gene from wild type.

Curr Biol, 1997 May 1, 7(5), 294 - 300
The protein kinase KSR interacts with 14-3-3 protein and Raf; Xing H et al.; BACKGROUND: KSR (kinase suppressor of Ras) is a recently identified putative protein kinase that positively mediates the Ras signaling pathway in the invertebrates Caenorhabditis elegans and Drosophila melanogaster . The function of vertebrate KSR is not well characterized biochemically or biologically . RESULTS: We examined the physiological role of KSR in vertebrate signal transduction using Xenopus laevis oocytes . Overexpression of KSR, in combination with overexpression of the intracellular dimeric protein 14-3-3, induced Xenopus oocyte meiotic maturation and cdc2 kinase activation; the effect of KSR and 14-3-3 on oocyte maturation was blocked by co-expression of dominant-negative Raf-1 . We noted that KSR contains multiple potential binding sites for 14-3-3, and we used the yeast two-hybrid system and co-immunoprecipitation experiments to show that KSR can bind to 14-3-3 . Furthermore, we demonstrated that KSR can form a complex with Raf kinase both in vitro and in cultured cells . Cell fractionation studies revealed that KSR formed a complex with 14-3-3 in both the membrane and cytoplasmic fractions of cell lysates; however, KSR only formed a complex with Raf-1 in the membrane fraction . CONCLUSIONS: Our finding suggest that KSR, 14-3-3 and Raf form an oligomeric signaling complex and that KSR positively regulates the Ras signaling pathway in vertebrate organisms.

Curr Biol, 1997 May 1, 7(5), R301 - 4
Motor proteins: myosin V--the multi-purpose transport motor; Titus MA; Studies in yeast and mice suggest that myosin V participates in the directed transport of a number of distinct cargos to polarized regions of the cell; myosin V has also been implicated in the provision of materials for filopodial extension in neurons.

Mol Cell Biol, 1997 May, 17(5), 2735 - 44
GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors; Hong H et al.; After binding to enhancer elements, transcription factors require transcriptional coactivator proteins to mediate their stimulation of transcription initiation . A search for possible coactivators for steroid hormone receptors resulted in identification of glucocorticoid receptor interacting protein 1 (GRIP1) . The complete coding sequence for GRIP1, isolated from a mouse brain cDNA library, contains an open reading frame of 1,462 codons . GRIP1 is the probable ortholog of the subsequently identified human protein transcription intermediary factor 2 (TIF2) and is also partially homologous to steroid receptor coactivator 1 (SRC-1) . The full-length GRIP1 interacted with the hormone binding domains (HBDs) of all five steroid receptors in a hormone-dependent manner and also with HBDs of class II nuclear receptors, including thyroid receptor alpha, vitamin D receptor, retinoic acid receptor alpha, and retinoid X receptor alpha . In contrast to agonists, glucocorticoid antagonists did not promote interaction between the glucocorticoid receptor and GRIP1 . In yeast cells, GRIP1 dramatically enhanced the transcriptional activation function of proteins containing the HBDs of any of the above-named receptors fused to the GAL4 DNA binding domain and thus served as a transcriptional coactivator for them . This finding contrasts with previous reports of TIF2 and SRC-1, which in mammalian cells enhanced the transactivation activities of only a subset of the steroid and nuclear receptors that they physically interacted with . GRIP1 also enhanced the hormone-dependent transactivation activity of intact glucocorticoid receptor, estrogen receptor, and mineralocorticoid receptor . Experiments with glucocorticoid receptor truncation and point mutants indicated that GRIP1 interacted with and enhanced the activity of the C-terminal AF-2 but not the N-terminal AF-1 transactivation domain of the glucocorticoid receptor . These results demonstrate directly that AF-1 and AF-2 domains accomplish their transactivation activities through different mechanisms: AF-2 requires GRIP1 as a coactivator, but AF-1 does not.

Mol Cell Biol, 1997 May, 17(5), 2679 - 87
RBP-L, a transcription factor related to RBP-Jkappa; Minoguchi S et al.; RBP-Jkappa is a sequence-specific DNA binding protein which plays a central role in signalling downstream of the Notch receptor by physically interacting with its intracellular region . Although at least four Notch genes exist in mammals, it is unknown whether each Notch requires a specific downstream signalling molecule . Here we report isolation and characterization of a mouse RBP-Jkappa-related gene named RBP-L that is expressed almost exclusively in lung, in contrast to the ubiquitous expression of RBP-Jkappa . For simplicity, we propose to call RBP-Jkappa RBP-J . The RBP-L protein bound to a DNA sequence almost identical to that of RBP-J . Surprisingly, RBP-L did not interact with any of the known four mouse Notch proteins . Although we found that RBP-L and EBNA-2 cooperated in transcriptional activation, they did not show significantly strong protein-protein interaction that can be detected by several in vivo and in vitro assays . This is again in contrast to physical association of RBP-J with EBNA-2 . Several models to explain functional interaction between RBP-L and EBNA-2 are discussed.

Mol Cell Biol, 1997 May, 17(5), 2669 - 78
Long-range interactions at the HO promoter; McBride HJ et al.; The SWI5 gene encodes a zinc finger DNA-binding protein required for the transcriptional activation of the yeast HO gene . There are two Swi5p binding sites in the HO promoter, site A at -1800 and site B at -1300 . Swi5p binding at site B has been investigated in some detail, and we have shown that Swi5p binds site B in a mutually cooperative fashion with Pho2p, a homeodomain protein . In this report, we demonstrate that Swi5p and Pho2p bind cooperatively to both sites A and B but that there are differences in binding to these two promoter sites . It has been shown previously that point mutations in either Swi5p binding site only modestly reduce HO expression in a PHO2 strain . We show that these mutant promoters are completely inactive in a pho2 mutant . We have created stronger point mutations at the two Swi5p binding sites within the HO promoter, and we show that the two binding sites, separated by 500 bp, are both absolutely required for HO expression, independent of PHO2 . These results create an apparent dilemma, as the strong mutations at the Swi5p binding sites show that both binding sites are required for HO expression, but the earlier binding site mutations allow Swi5p to activate HO, but only in the presence of Pho2p . To explain these results, a model is proposed in which physical interaction between Swi5p proteins bound to these two sites separated by 500 bp is required for activation of the HO promoter . Experimental evidence is presented that supports the model . In addition, through deletion analysis we have identified a region near the amino terminus of Swi5p that is required for PHO2-independent activation of HO, suggesting that this region mediates the long-range interactions between Swi5p molecules bound at the distant sites.

Mol Cell Biol, 1997 May, 17(5), 2538 - 49
The DNA binding and activation domains of Gal4p are sufficient for conveying its regulatory signals; Ding WV et al.; The transcriptional activation function of the Saccharomyces cerevisiae activator Gal4p is known to rely on a DNA binding activity at its amino terminus and an activation domain at its carboxy terminus . Although both domains are required for activation, truncated forms of Gal4p containing only these domains activate poorly in vivo . Also, mutations in an internal conserved region of Gal4p inactivate the protein, suggesting that this internal region has some function critical to the activity of Gal4p . We have addressed the question of what is the minimal form of Gal4 protein that can perform all of its known functions . A form with an internal deletion of the internal conserved domain of Gal4p is transcriptionally inactive, allowing selection for suppressors . All suppressors isolated were intragenic alterations that had further amino acid deletions (miniGAL4s) . Characterization of the most active miniGal4 proteins demonstrated that they possess all of the known functions of full-length Gal4p, including glucose repression, galactose induction, response to deletions of gal11 or gal6, and interactions with other proteins such as Ga180p, Sug1p, and TATA binding protein . Analysis of the transcriptional activities, protein levels, and DNA binding abilities of these miniGal4ps and a series of defined internal mutants compared to those of the full-length Gal4p indicates that the DNA binding and activation domains are necessary and sufficient qualitatively for all of these known functions of Gal4p . Our observations imply that the internal region of Gal4 protein may serve as a spacer to augment transcription and/or may be involved in intramolecular or Gal4p-Gal4p interactions.

Mol Cell Biol, 1997 May, 17(5), 2436 - 47
Genetic and biochemical analysis of Msh2p-Msh6p: role of ATP hydrolysis and Msh2p-Msh6p subunit interactions in mismatch base pair recognition; Alani E et al.; Recent studies have shown that Saccharomyces cerevisiae Msh2p and Msh6p form a complex that specifically binds to DNA containing base pair mismatches . In this study, we performed a genetic and biochemical analysis of the Msh2p-Msh6p complex by introducing point mutations in the ATP binding and putative helix-turn-helix domains of MSH2 . The effects of these mutations were analyzed genetically by measuring mutation frequency and biochemically by measuring the stability, mismatch binding activity, and ATPase activity of msh2p (mutant msh2p)-Msh6p complexes . A mutation in the ATP binding domain of MSH2 did not affect the mismatch binding specificity of the msh2p-Msh6p complex; however, this mutation conferred a dominant negative phenotype when the mutant gene was overexpressed in a wild-type strain, and the mutant protein displayed biochemical defects consistent with defects in mismatch repair downstream of mismatch recognition . Helix-turn-helix domain mutant proteins displayed two different properties . One class of mutant proteins was defective in forming complexes with Msh6p and also failed to recognize base pair mismatches . A second class of mutant proteins displayed properties similar to those observed for the ATP binding domain mutant protein . Taken together, these data suggested that the proposed helix-turn-helix domain of Msh2p was unlikely to be involved in mismatch recognition . We propose that the MSH2 helix-turn-helix domain mediates changes in Msh2p-Msh6p interactions that are induced by ATP hydrolysis; the net result of these changes is a modulation of mismatch recognition.

Mol Cell Biol, 1997 May, 17(5), 2353 - 9
Function of the c-Myc antagonist Mad1 during a molecular switch from proliferation to differentiation; Cultraro CM et al.; Mad-Max heterodimers have been shown to antagonize Myc transforming activity by a mechanism requiring multiple protein-protein and protein-DNA interactions . However, the mechanism by which Mad functions in differentiation is unknown . Here, we present evidence that Mad functions by an active repression mechanism to antagonize the growth-promoting function(s) of Myc and bring about a transition from cellular proliferation to differentiation . We demonstrate that exogenously expressed c-Myc blocks inducer-mediated differentiation of murine erythroleukemia cells without disrupting the induction of endogenous Mad; rather, high levels of c-Myc prevent a heterocomplex switch from growth-promoting Myc-Max to growth-inhibitory Mad-Max . Cotransfection of a constitutive c-myc with a zinc-inducible mad1 results in clones expressing both genes, whereby a switch from proliferation to differentiation can be modulated . Whereas cells grown in N'N'-hexamethylene bisacetamide in the absence of zinc fail to differentiate, addition of zinc up-regulates Mad expression by severalfold and differentiation proceeds normally . Coimmunoprecipitation analysis reveals that Mad-Max complexes are in excess of Myc-Max in these cotransfectants . Moreover, we show that the Sin-binding, basic region, and leucine zipper motifs are required for Mad to function during a molecular switch from proliferation to differentiation.

J Neurobiol, 1997 May, 32(5), 443 - 56
Neuroblast ablation in Drosophila P{GAL4} lines reveals origins of olfactory interneurons; Stocker RF et al.; Hydroxyurea (HU) treatment of early first instar larvae in Drosophila was previously shown to ablate a single dividing lateral neuroblast (LNb) in the brain . Early larval HU application to P{GAL4} strains that label specific neuron types enabled us to identify the origins of the two major classes of interneurons in the olfactory system . HU treatment resulted in the loss of antennal lobe local interneurons and of a subset of relay interneurons (RI), elements usually projecting to the calyx and the lateral protocerebrum (LPR) . Other RI were resistant to HU and still projected to the LPR . However, they formed no collaterals in the calyx region (which was also ablated), suggesting that their survival does not depend on targets in the calyx . Hence, the ablated interneurons were derived from the LNb, whereas the HU-resistant elements originated from neuroblasts which begin to divide later in larval life . Developmental GAL4 expression patterns suggested that differentiated RI are present at the larval stage already and may be retained through metamorphosis.

Nucleic Acids Res, 1997 May 1, 25(9), 1825 - 9
DNA polymerization catalysed by a group II intron RNA in vitro; Hetzer M et al.; The excised group II intron bI1 from Saccharomyces cerevisiae can act as a ribozyme catalysing various chemical reactions with different substrate RNAs in vitro . Recently, we have described an editing-like RNA polymerization reaction catalysed by the bI1 intron lariat that proceeds in the 3'-->5'direction . Here we show that the bI1 lariat RNA can also catalyse successive deoxyribonucleotide polymerization reactions on exogenous substrate molecules . The basic mechanism of the reaction involved interacting cycles between an alternative version of partial reverse splicing (lariat charging) and canonical forward splicing (lariat discharging by exon ligation) . With an overall chain growth in the 3'-->5' direction, the 5' exon RNAs (IBS1dN) were elongated by successive insertion of deoxyribonucleotides derived from single deoxyribonucleotide substitutions (dA, dG, dC or dT) . All four deoxyribonucleotides were used as substrates, although with different efficiencies . Our findings extend the catalytic repertoire of group II intron RNAs not only by a novel DNA polymerization activity, but also by a DNA-DNA ligation capacity, supporting the idea that ribozymes might have been part of the first primordial polymerization machinery for both RNA and DNA.

Mol Cells, 1997 Apr 30, 7(2), 220 - 5
Rapid analysis for the isolation of novel genes encoding putative effectors to the position-specific regulatory element of murine Hoxa-7; Cho M et al.; Hox genes are known to play a critical role in pattern formation during vertebrate development by being expressed at the specific time and in the specific position along the antero-posterior body axis . In order to understand the regulatory mechanism for the position-specific expression of murine Hoxa-7, yeast one-hybrid system was applied . DNA fragment conferring a position specificity to the Hoxa-7 gene was placed just upstream from the yeast CYC1 promoter and lacZ gene in a reporter . Selection of LacZ positive clones after cotransformation of the reporter and mouse embryonic cDNA library as an effector, which was designed to be expressed as fusion proteins to the GAL4 activation domain, allowed us to isolate putative factors interacting with the position-specific regulatory element of murine Hoxa-7 . A total of 28 positive clones were screened from 5 x 10(5) yeast transformants . About 70% of the clones turned out to be novel and most of the candidate clones selected in this study showed a temporally restricted expression pattern during embryonic development, suggesting that this method could provide an efficient way for isolating novel genes whose expressions are temporally regulated during embryogenesis.

Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4289 - 94
Gene induction in response to unfolded protein in the endoplasmic reticulum is mediated through Ire1p kinase interaction with a transcriptional coactivator complex containing Ada5p; Welihinda AA et al.; In eukaryotic cells, accumulation of unfolded protein in the endoplasmic reticulum induces transcription of a family of genes encoding endoplasmic reticulum protein chaperones through a conserved unfolded protein response element . In Saccharomyces cerevisiae, activation of a transmembrane receptor kinase, Ire1p (Ern1p), initiates signaling, although the mediators immediately downstream of Ire1 kinase are unknown . Here we demonstrate interaction of Ire1p with the transcriptional coactivator, Gcn5p (for general control nonrepressed; also known as Ada4p) . Gcn5p associates with other Ada (for alteration/deficiency in activation) gene products in a heteromeric complex and has histone acetyltransferase activity . We show that the Gcn5/Ada complex is selectively required for the unfolded protein response but not for the heat shock response . A novel mechanism is proposed in which activation of a receptor kinase recruits a transcription coactivator complex to a specific chromosomal locus to mediate localized histone acetylation, thus making specific gene sequences accessible for transcription.

Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4267 - 72
DNA looping by Ku and the DNA-dependent protein kinase; Cary RB et al.; The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription . The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70/86 heterodimer and an approximately 460-kDa catalytic subunit (DNA-PKcs) . DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair . Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and protein-nucleic acid complexes without staining or metal coating . The AFM provides a rapid and direct means of probing the protein-nucleic acid interactions responsible for DNA repair and genetic regulation . Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA . A significant number of DNA molecules formed loops in the presence of Ku . DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs . Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates . We conclude that, when bound to DNA, Ku is capable of self-association . These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.

Biochem Biophys Res Commun, 1997 Apr 28, 233(3), 765 - 9
ERC-55, a binding protein for the papilloma virus E6 oncoprotein, specifically interacts with vitamin D receptor among nuclear receptors; Imai T et al.; VDR regulates gene expression in a ligand-dependent way by binding to cognate enhancer elements of target gene promoters . The ligand-dependent activation function, AF-2, of VDR is thought to require transcriptional co-activators/co-repressors together with basal transcriptional machinery . Using a yeast two hybrid system with VDR, we have isolated a mouse Ca(2+)-binding protein (designated as VAF1) specifically interacting in vivo and in vitro with VDR among nuclear receptors like RAR, RXR, ER and GR . VAF1 is a mouse homologue to human ERC-55, which has recently been shown to interact with human papillomavirus oncogenic protein, E6{1} . Unlike those of many previously identified co-activators, the VDR-VAF1 interaction was ligand-independent . Thus, VAF1 seems a putative VDR-specific cofactor modulating its function.

FEBS Lett, 1997 Apr 28, 407(2), 220 - 4
The protein encoded by the MFT1 gene is a targeting factor for mitochondrial precursor proteins, and not a core ribosomal protein; Beilharz T et al.; Yeast cells harboring mft1 mutations are compromised in mitochondrial protein targeting, and Mft1p has previously been identified as a ribosomal protein . However, two genes, PLC2 and YML062C, are present in the MFT1 locus, and we show that mft1 mutant cells are compromised in the function of the cytosolic protein encoded by YML062C . The ribosomal protein (YS3a) is actually encoded by the tightly linked PLC2 gene, and does not play a role in targeting proteins to the mitochondria.

J Biol Chem, 1997 Apr 25, 272(17), 11215 - 20
Regulation of phospholipid biosynthetic enzymes by the level of CDP-diacylglycerol synthase activity; Shen H et al.; Amine-containing phospholipid synthesis in Saccharomyces cerevisiae starts with the conversion of CDP-diacylglycerol (CDP-DAG) and serine to phosphatidylserine (PS), whereas phosphatidylinositol (PI) is formed from CDP-DAG and inositol (derived from inositol 1-phosphate) . In this study the regulation of PS synthase (encoded by CHO1/PSS), PI synthase (encoded by PIS1), and inositol 1-phosphate synthase (encoded by INO1) activities by the in vivo level of CDP-DAG synthase activity (encoded by CDS1) is described . Reduction in the level of CDP-DAG synthase activity from 10-fold over wild type levels to 10% of wild type levels results in a 7-fold increase in PS synthase activity, which follows a similar change in the CHO1/PSS mRNA level . INO1 mRNA also increases but only after CDP-DAG synthase activity falls below the wild type level . PI synthase activity follows the decrease of the CDP-DAG synthase activity, but there is no parallel change in the level of PIS1 mRNA . These changes in CHO1/PSS and INO1 mRNA levels are mediated by a mechanism not dependent on changes in the expression of the INO2-OPI1 regulatory genes . CDS1 expression is repressed in concert with INO2 expression in response to inositol.

J Biol Chem, 1997 Apr 25, 272(17), 11205 - 14
Activation of hypoxia-inducible factor-1; definition of regulatory domains within the alpha subunit; Pugh CW et al.; Hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins (HIF-1alpha and -1beta), is a key component of a widely operative transcriptional response activated by hypoxia, cobaltous ions, and iron chelation . To identify regions of HIF-1 subunits responsible for oxygen-regulated activity, we constructed chimeric genes in which portions of coding sequence from HIF-1 genes were either linked to a heterologous DNA binding domain or encoded between such a DNA binding domain and a constitutive activation domain . Sequences from HIF-1alpha but not HIF-1beta conferred oxygen-regulated activity . Two minimal domains within HIF-1alpha (amino acids 549-582 and amino acids 775-826) were defined by deletional analysis, each of which could act independently to convey inducible responses . Both these regions confer transcriptional activation, and in both cases adjacent sequences appeared functionally repressive in transactivation assays . The inducible operation of the first domain, but not the second, involved major changes in the level of the activator fusion protein in transfected cells, inclusion of this sequence being associated with a marked reduction of expressed protein level in normoxic cells, which was relieved by stimulation with hypoxia, cobaltous ions, or iron chelation . These results lead us to propose a dual mechanism of activation in which the operation of an inducible activation domain is amplified by regulation of transcription factor abundance, most likely occurring through changes in protein stability.

J Biol Chem, 1997 Apr 25, 272(17), 11193 - 7
A complex composed of tup1 and ssn6 represses transcription in vitro; Redd MJ et al.; The Saccharomyces cerevisiae Tup1 protein is a member of a family of WD repeat containing proteins that are involved in repression of transcription . Tup1, along with the Ssn6 protein, represses a wide variety of genes in yeast including cell type-specific and glucose-repressed genes . Tup1 and Ssn6 are recruited to these specific gene sets by interaction with sequence-specific DNA binding proteins . In this work, a protein complex containing Ssn6 and Tup1 was purified to determine its composition . The size of the complex is estimated to be 440 kDa . Tup1 and Ssn6, which are both phosphoproteins, are the only proteins present in stoichiometric amounts in the complex . We also demonstrate that this purified complex represses transcription in an in vitro assay.

Science, 1997 Apr 25, 276(5312), 614 - 7
Continuous in vitro evolution of catalytic function; Wright MC et al.; A population of RNA molecules that catalyze the template-directed ligation of RNA substrates was made to evolve in a continuous manner in the test tube . A simple serial transfer procedure was used to achieve approximately 300 successive rounds of catalysis and selective amplification in 52 hours . During this time, the population size was maintained against an overall dilution of 3 x 10(298) . Both the catalytic rate and amplification rate of the RNAs improved substantially as a consequence of mutations that accumulated during the evolution process . Continuous in vitro evolution makes it possible to maintain laboratory "cultures" of catalytic molecules that can be perpetuated indefinitely.

Science, 1997 Apr 25, 276(5312), 561 - 7
Reverse transcriptase motifs in the catalytic subunit of telomerase; Lingner J et al.; Telomerase is a ribonucleoprotein enzyme essential for the replication of chromosome termini in most eukaryotes . Telomerase RNA components have been identified from many organisms, but no protein component has been demonstrated to catalyze telomeric DNA extension . Telomerase was purified from Euplotes aediculatus, a ciliated protozoan, and one of its proteins was partially sequenced by nanoelectrospray tandem mass spectrometry . Cloning and sequence analysis of the corresponding gene revealed that this 123-kilodalton protein (p123) contains reverse transcriptase motifs . A yeast (Saccharomyces cerevisiae) homolog was found and subsequently identified as EST2 (ever shorter telomeres), deletion of which had independently been shown to produce telomere defects . Introduction of single amino acid substitutions within the reverse transcriptase motifs of Est2 protein led to telomere shortening and senescence in yeast, indicating that these motifs are important for catalysis of telomere elongation in vivo . In vitro telomeric DNA extension occurred with extracts from wild-type yeast but not from est2 mutants or mutants deficient in telomerase RNA . Thus, the reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.

Oncogene, 1997 Apr 24, 14(16), 1999 - 2004
Interaction between Cdc37 and Cdk4 in human cells; Lamphere L et al.; Using the yeast two-hybrid system we have identified novel potential Cdk4 interacting proteins . Here we described the interaction of Cdk4 with a human homologue of the yeast Drosophila CDC37 gene products . Cdc37 protein specifically interacts with Cdk4 and Cdk6, but not with Cdc2, Cdk2, Cdk3, Cdk5 and any of a number of cyclins tested . Cdc37 is not an inhibitor nor an activator of the Cdk4/cyclin D1 kinase, while it appears to facilitate complex assembly between Cdk4, and cyclin D1 in vitro . Cdc37 competes with p16 for binding to Cdk4, suggesting that p16 might exert part of its inhibitory function by affecting the formation of Cdk4/cyclin D1 complexes via Cdc37.

Nature, 1997 Apr 24, 386(6627), 804 - 10
Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2; Sharan SK et al.; Inherited mutations in the human BRCA2 gene cause about half of the cases of early-onset breast cancer . The embryonic expression pattern of the mouse Brca2 gene is now defined and an interaction identified of the Brca2 protein with the DNA-repair protein Rad51 . Developmental arrest in Brca2-deficient embryos, their radiation sensitivity, and the association of Brca2 with Rad51 indicate that Brca2 may be an essential cofactor in the Rad51-dependent DNA repair of double-strand breaks, thereby explaining the tumour-suppressor function of Brca2.

J Cell Biol, 1997 Apr 21, 137(2), 377 - 86
Tim23, a protein import component of the mitochondrial inner membrane, is required for normal activity of the multiple conductance channel, MCC; Lohret TA et al.; We previously showed that the conductance of a mitochondrial inner membrane channel, called MCC, was specifically blocked by peptides corresponding to mitochondrial import signals . To determine if MCC plays a role in protein import, we examined the relationship between MCC and Tim23p, a component of the protein import complex of the mitochondrial inner membrane . We find that antibodies against Tim23p, previously shown to inhibit mitochondrial protein import, inhibit MCC activity . We also find that MCC activity is altered in mitochondria isolated from yeast carrying the tim23-1 mutation . In contrast to wild-type MCC, we find that the conductance of MCC from the tim23-1 mutant is not significantly blocked by mitochondrial presequence peptides . Tim23 antibodies and the tim23-1 mutation do not, however, alter the activity of PSC, a presequence-peptide sensitive channel in the mitochondrial outer membrane . Our results show that Tim23p is required for normal MCC activity and raise the possibility that precursors are translocated across the inner membrane through the pore of MCC.

Cell, 1997 Apr 18, 89(2), 195 - 204
Disruption of mouse RAD54 reduces ionizing radiation resistance and homologous recombination; Essers J et al.; Double-strand DNA break (DSB) repair by homologous recombination occurs through the RAD52 pathway in Saccharomyces cerevisiae . Its biological importance is underscored by the conservation of many RAD52 pathway genes, including RAD54, from fungi to humans . We have analyzed the phenotype of mouse RAD54-/- (mRAD54-/-) cells . Consistent with a DSB repair defect, these cells are sensitive to ionizing radiation, mitomycin C, and methyl methanesulfonate, but not to ultraviolet light . Gene targeting experiments demonstrate that homologous recombination in mRAD54-/- cells is reduced compared to wild-type cells . These results imply that, besides DNA end-joining mediated by DNA-dependent protein kinase, homologous recombination contributes to the repair of DSBs in mammalian cells . Furthermore, we show that mRAD54-/- mice are viable and exhibit apparently normal V(D)J and immunoglobulin class-switch recombination . Thus, mRAD54 is not required for the recombination processes that generate functional immunoglobulin and T cell receptor genes.

Cell, 1997 Apr 18, 89(2), 185 - 93
Reduced X-ray resistance and homologous recombination frequencies in a RAD54-/- mutant of the chicken DT40 cell line; Bezzubova O et al.; rad54 mutants of the yeast Saccharomyces cerevisiae are extremely X-ray sensitive and have decreased mitotic recombination frequencies because of a defect in double-strand break repair . A RAD54 homolog was disrupted in the chicken B cell line DT40, which undergoes immunoglobulin gene conversion and exhibits unusually high ratios of targeted to random integration after DNA transfection . Homozygous RAD54-/- mutant clones were highly X-ray sensitive compared to wildtype cells . The rate of immunoglobulin gene conversion was 6- to 8-fold reduced, and the frequency of targeted integration was at least two orders of magnitude decreased in the mutant clones . Reexpression of the RAD54 cDNA restored radiation resistance and targeted integration activity . The reported phenotype provides the first genetic evidence of a link between double-strand break repair and homologous recombination in vertebrate cells.

J Biol Chem, 1997 Apr 18, 272(16), 10797 - 803
Interferon-gamma activation of a mitogen-activated protein kinase, KFR1, in the bloodstream form of Trypanosoma brucei; Hua SB et al.; KFR1, a mitogen-activated protein (MAP) kinase identified in the African trypanosome, Trypanosoma brucei, is a serine protein kinase capable of phosphorylating the serine residues in histone H-1, myelin basic protein, and beta-casein . It phosphorylates four proteins with estimated molecular masses of 22, 34, 46, and 90 kDa from the T . brucei bloodstream-form lysate in vitro . KFR1 bears significant sequence similarity to the yeast MAP kinases KSS1 and FUS3 but cannot functionally complement the kss1/fus3 yeast mutant . It is encoded by a single-copy gene in the diploid T . brucei, and only one of the two alleles can be successfully disrupted, suggesting an essential function of KFR1 in T . brucei . KFR1 activity is present at a much enhanced level in the bloodstream form of T . brucei when compared with that in the insect (procyclic) form . This enhanced activity can be eliminated in vitro by the treatment with protein phosphatase HVH2 known to act specifically on MAP kinases . It can also be decreased in the bloodstream form of T . brucei by serum starvation but induced specifically by interferon-gamma . The production of interferon-gamma in the mammalian host is known to be triggered by T . brucei infection, and this cytokine, as has been reported, promotes the proliferation of T . brucei in the mammalian blood . Since none of these phenomena can be observed in the procyclic form of T . brucei, activation of KFR1 is most likely involved in mediating the interferon-gamma-induced proliferation of T . brucei in the mammalian host.

J Biol Chem, 1997 Apr 18, 272(16), 10704 - 9
Translocation of rhoA associated with Ca2+ sensitization of smooth muscle; Gong MC et al.; We determined the relationship between the localization of rhoA and Ca2+ sensitization of force in smooth muscle . In alpha-toxin-permeabilized rabbit portal vein at pCa 6.5, the particulate hydrophobic fraction of rhoA (10 +/- 1.6% of the total) was significantly increased by phenylephrine to 18 +/- 5.5% at 5 min, by AlF4- to 26 +/- 8.4% at 20 min, and dose-dependently up to 62 +/- 9.5% by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS; 0.3-50 microM) . Translocation of rhoA was selective (Rac1 and Cdc42 were not translocated) and was quantitatively correlated (up to approximately 50%; r = 0.91, p < 0.05) with Ca2+ sensitization; high GTPgammaS concentrations (>/=10 microM) further increased translocation without increasing force . The initial recruitment of rhoA to the membrane paralleled the time course of contraction, but sensitization could be reversed without a decrease in particulate rhoA . High {Ca2+} (pCa 4.5) also increased particulate rhoA to 31 +/- 5.8% . Membrane-associated rhoA in unstimulated portal vein was a good substrate for in vitro ADP-ribosylation, whereas the large amount translocated by GTPgammaS was not . We conclude that 1) translocation of rhoA plays a causal role in Ca2+ sensitization, and 2) membrane-bound rhoA can exist in two or more states.

J Biol Chem, 1997 Apr 18, 272(16), 10498 - 505
Tandem duplication of rab genes followed by sequence divergence and acquisition of distinct functions in Trypanosoma brucei; Field H et al.; The Ras superfamily of small G proteins governs unidirectional cellular processes by virtue of GTP hydrolysis and concomitant conformational changes, which are in turn regulated by a number of accessory factors . Members of the Rab subfamily are important for correct targeting and fusion of intra-organellar vesicles loaded with trafficking proteins and lipids . During evolution from a prototype gene, novel functions may be acquired by duplicated daughter genes; for Rab proteins, this can be tested by location, which is specifically related to the function of each Rab . We have found an example of two rab genes in Trypanosoma brucei (trab genes) that clearly arose by tandem duplication, being highly related to each other and remaining juxtaposed in the genome, whose products have dramatically different subcellular locations, indicative of discrete functions . These two trab genes, isolated on a single genomic clone, are separated by a short intervening sequence and are in a head-to-tail orientation . The nucleotide sequences of the open reading frames and intervening sequence were determined and show that the genes are paralogues, probably arising from an ancient tandem duplication . Both genes are most homologous to ypt1 and sec4 in the Saccharomyces cerevisiae genome, while phylogenetic reconstruction indicates that although they have clearly diverged, the proteins are more closely related to each other than to other Rab protein sequences available in the data base . Immunofluorescence microscopy, using antibodies raised against the recombinant Trab proteins, clearly demonstrates that the native Trab proteins have completely distinct subcellular locations in the trypanosome . Trab1p is present in a widespread reticular location similar to BiP, suggesting an endoplasmic reticulum location, while Trab7p is observed in a discrete structure adjacent to the kinetoplast . Most interestingly, the Trab7p-positive compartment also appears to divide at the same time, or just prior to, the kinetoplast, i.e . early in mitosis, suggestive of association with structures in the flagellar pocket region . An estimate of the divergence time indicates that the trab1/trab7 duplication occurred approximately 100 million years ago, and therefore, the persistence of this pair suggests an essential role in the survival of T . brucei.

Biochem Biophys Res Commun, 1997 Apr 17, 233(2), 492 - 5
Nitric oxide-releasing particles inhibit phagocytosis in human neutrophils; Forslund T et al.; We have constructed a yeast (Saccharomyces cerevisiae) particle capable of releasing NO, by loading heat-killed yeast particles with a hydrophobic NO-generating substance, GEA-5171 . This particle decreased phagocytosis in solution, as measured with flow cytometry, to about 80% of control values . Phagocytosis on a surface, as counted under the microscope, was also decreased by about 20% . The nitric oxide furthermore counteracted the production of oxygen metabolites by neutrophils to about 20% of control values . The inhibitory effect was most pronounced for the intracellular production, as could be seen when neutrophils preincubated with NO-releasing particles were stimulated with chemotactic agent (FMLP) or phorbol ester (PMA) . In conclusion, NO has inhibitory effects on both phagocytosis and the respiratory burst of neutrophils . Since nitric oxide is a hydrophobic gas and an air pollutant, there is a possibility that it accumulates in particles which then become more resistant to elimination.

Biochem Biophys Res Commun, 1997 Apr 17, 233(2), 343 - 8
Specific cleavage of the large subunit of replication factor C in apoptosis is mediated by CPP32-like protease; Song Q et al.; Recent evidence suggests that the growing family of cysteine proteases related to the interleukin-1beta-converting enzyme (ICE) is of central importance in mediating apoptosis . Proteolytic cleavage of a small group of cellular substrates by these enzymes in association with the onset of apoptosis has been reported . In the present study, we searched a protein data base for potential death substrates possessing the CPP32 cleavage site, DEVD, and identified several candidates including RFC140, the large subunit of replication factor C, which we subsequently demonstrated to be specifically cleaved in a variety of cell types undergoing apoptosis in response to different cytotoxic agents, whereas no degradation is observed in a cell line resistant to etoposide-induced apoptosis . The abrogation of RFC140 cleavage in apoptotic extracts by Ac-DEVD-CHO, a potent inhibitor of CPP32, together with the finding that a CPP32 consensus cleavage sequence, DEVD, exists in RFC140, suggests that CPP32 or a close relative is responsible for RFC140 degradation in apoptosis.

Nature, 1997 Apr 17, 386(6626), 732 - 5
Functional interaction between DNA-PK and c-Abl in response to DNA damage; Kharbanda S et al.; How DNA damage is converted into intracellular signals that can control cell behaviour is unknown . The c-Abl protein tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents, whereas the DNA-dependent protein kinase (DNA-PK), consisting of a serine/threonine kinase and Ku DNA-binding subunits, requires DNA double-strand breaks or other DNA lesions for activation . Here we demonstrate that c-Abl interacts constitutively with DNA-PK . Ionizing radiation stimulates binding of c-Abl to DNA-PK and induces an association of c-Abl with Ku antigen . We show that DNA-PK phosphorylates and activates c-Abl in vitro . Cells deficient in DNA-PK are defective in c-Abl activation induced by ionizing radiation . In a potential feedback mechanism, c-Abl phosphorylates DNA-PK, but not Ku, in vitro . Phosphorylation of DNA-PK by c-Abl inhibits the ability of DNA-PK to form a complex with DNA . We also show that treatment of cells with ionizing radiation results in phosphorylation of DNA-PK that is dependent on c-Abl . Our results support the hypothesis that there are functional interactions between c-Abl and DNA-PK in the response to DNA damage.

EMBO J, 1997 Apr 15, 16(8), 2150 - 60
Chromatin structure modulates DNA repair by photolyase in vivo; Suter B et al.; Yeast and many other organisms use nucleotide excision repair (NER) and photolyase in the presence of light (photoreactivation) to repair cyclobutane pyrimidine dimers (CPDs), a major class of DNA lesions generated by UV light . To study the role of photoreactivation at the chromatin level in vivo, we used yeast strains which contained minichromosomes (YRpTRURAP, YRpCS1) with well-characterized chromatin structures . The strains were either proficient (RAD1) or deficient (rad1 delta) in NER . In contrast to NER, photolyase rapidly repairs CPDs in non-nucleosomal regions, including promoters of active genes (URA3, HIS3, DED1) and in linker DNA between nucleosomes . CPDs in nucleosomes are much more resistant to photoreactivation . These results demonstrate a direct role of chromatin in modulation of a DNA repair process and an important role of photolyase in repair of damaged promoters with presumptive effects on gene regulation . In addition, photoreactivation provides an in vivo test for chromatin structure and stability . In active genes (URA3, HIS3), photolyase repairs the non-transcribed strand faster than the transcribed strand and can match fast removal of lesions from the transcribed strand by NER (transcription-coupled repair) . Thus, the combination of both repair pathways ensures efficient repair of active genes.

EMBO J, 1997 Apr 15, 16(8), 2096 - 107
Histone acetylation: influence on transcription, nucleosome mobility and positioning, and linker histone-dependent transcriptional repression; Ura K et al.; We demonstrate using a dinucleosome template that acetylation of the core histones enhances transcription by RNA polymerase III . This effect is not dependent on an increased mobility of the core histone octamer with respect to DNA sequence . When linker histone is subsequently bound, we find both a reduction in nucleosome mobility and a repression of transcription . These effects of linker histone binding are independent of core histone acetylation, indicating that core histone acetylation does not prevent linker histone binding and the concomitant transcriptional repression . These studies are complemented by the use of a Xenopus egg extract competent both for chromatin assembly on replicating DNA and for RNA polymerase III transcription . Incorporation of acetylated histones and lack of linker histones together facilitate transcription by >10-fold in this system; however, they have little independent effect on transcription . Thus core histone acetylation significantly facilitates transcription, but this effect is inhibited by the assembly of linker histones into chromatin.

EMBO J, 1997 Apr 15, 16(8), 1934 - 42
G proteins in Ustilago maydis: transmission of multiple signals?
Regenfelder E, Spellig T, Hartmann A, Lauenstein S, Bolker M, Kahmann R.
In the phytopathogenic fungus Ustilago maydis, cell fusion is governed by a pheromone signalling system . The pheromone receptors belong to the seven transmembrane class that are coupled to heterotrimeric G proteins . We have isolated four genes (gpa1 to gpa4) encoding alpha subunits of G proteins . Gpa1, Gpa2 and Gpa3 have homologues in other fungal species, while Gpa4 is novel . Null mutants in individual genes were viable and only disruption of gpa3 caused a discernible phenotype . gpa3 mutant strains were unable to respond to pheromone and thus were mating-deficient . A constitutively active allele of gpa3 (gpa3(Q206L)) was generated by site-directed mutagenesis . Haploid strains harbouring gpa3(Q206L) were able to mate without pheromone stimulation, indicating that Gpa3 plays an active role in transmission of the pheromone signal . Surprisingly, Gpa3 is also required for pathogenic development, although pheromone signalling is not essential for this process.

EMBO J, 1997 Apr 15, 16(8), 1876 - 87
Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1; Egloff MP et al.; The diverse forms of protein phosphatase 1 in vivo result from the association of its catalytic subunit (PP1c) with different regulatory subunits, one of which is the G-subunit (G(M)) that targets PP1c to glycogen particles in muscle . Here we report the structure, at 3.0 A resolution, of PP1c in complex with a 13 residue peptide (G(M{63-75})) of G(M) . The residues in G(M{63-75}) that interact with PP1c are those in the Arg/Lys-Val/Ile-Xaa-Phe motif that is present in almost every other identified mammalian PP1-binding subunit . Disrupting this motif in the G(M{63-75}) peptide and the M(110{1-38}) peptide (which mimics the myofibrillar targeting M110 subunit in stimulating the dephosphorylation of myosin) prevents these peptides from interacting with PP1 . A short peptide from the PP1-binding protein p53BP2 that contains the RVXF motif also interacts with PP1c . These findings identify a recognition site on PP1c, invariant from yeast to humans, for a critical structural motif on regulatory subunits . This explains why the binding of PP1 to its regulatory subunits is mutually exclusive, and suggests a novel approach for identifying the functions of PP1-binding proteins whose roles are unknown.

EMBO J, 1997 Apr 15, 16(8), 1842 - 9
Sequential action of two hsp70 complexes during protein import into mitochondria; Horst M et al.; The mitochondrial chaperone mhsp70 mediates protein transport across the inner membrane and protein folding in the matrix . These two reactions are effected by two different mhsp70 complexes . The ADP conformation of mhsp70 favors formation of a complex on the inner membrane; this 'import complex' contains mhsp70, its membrane anchor Tim44 and the nucleotide exchange factor mGrpE . The ATP conformation of mhsp70 favors formation of a complex in the matrix; this 'folding complex' contains mhsp70, the mitochondrial DnaJ homolog Mdj1 and mGrpE . A precursor protein entering the matrix interacts first with the import complex and then with the folding complex . A chaperone can thus function as part of two different complexes within the same organelle.

Eur J Biochem, 1997 Apr 15, 245(2), 241 - 51
Characterization of the basal and pheromone-stimulated phosphorylation states of Ste12p; Hung W et al.; The Saccharomyces cerevisiae transcription factor Ste12p is required for basal and activated expression of pheromone-responsive genes, and for invasive growth in haploid cells . In diploid yeast, Ste12p is implicated in pseudohyphal development . The ability of Ste12p to effect these various responses in three different cell types must require stringent regulation of its transcriptional activation function and interaction with additional transcription factors . We have examined the phosphorylation state of Ste12p in untreated and pheromone-treated haploid cells, and found eight constitutively phosphorylated peptides . Phosphorylation at the constitutive sites does not require the protein kinases of the pheromone-response pathway . Treatment of haploid yeast with mating pheromone causes the appearance of novel relatively minor phosphorylations on Ste12p . Brief {35S}methionine labeling reveals novel pheromone-dependent, electrophoretically slower migrating Ste12p species . Similarly, the sole difference we observe in tryptic phosphopeptides generated from Ste12p from pheromone-treated and untreated cells is the transient appearance of two novel minor hydrophobic phosphopeptides . The pheromone-dependent phosphorylation of Ste12p requires an intact pheromone-response pathway and localization of Ste12p to the nucleus, but does not require the Ste12p DNA-binding domain . We conclude from these experiments that the pheromone-response pathway induces the formation of specific hyperphosphorylation on Ste12p, which can only be detected as apparently minor modifications in vivo . We argue that, if Ste12p is regulated by direct pheromone-responsive phosphorylation, then that phosphorylation must be represented by the two novel phosphopeptides . However, we cannot exclude the possibility that pheromone-responsive transcription is controlled by direct phosphorylation of a target other than Ste12p.

Arch Biochem Biophys, 1997 Apr 15, 340(2), 201 - 7
Molecular cloning and characterization of a novel putative STE20-like kinase in guinea pigs; Itoh S et al.; Protein kinases play a key role in cell growth and differentiation . We have isolated the cDNA of a novel protein serine/threonine kinase (referred to as STE20-like kinase (SLK) from a guinea pig liver cDNA library with a probe generated by a cloning approach based on the polymerase chain reaction . The encoded polypeptide (1231 amino acids, M(r) 141,079) contains all conserved subdomains characteristic of the protein serine threonine kinase family . A hemagglutinin-tagged SLK expressed artificially in COS7 cells was hyperphosphorylated by anisomycin . By Northern blot analysis, SLK mRNA was detected in all organs examined: brain, lung heart, liver, kidney, spleen, testis, and eosinophils . Sequence comparisons of its catalytic domain related SLK to p21-activated kinase family of protein serine/threonine kinases . Its noncatalytic domain comprises several intriguing structural features, including the acidic region and the nuclear targeting sequence . This noncatalytic domain exhibited no extended similarity with other proteins . Thus, SLK is a protein serine/threonine kinase which contains an unknown regulatory domain(s).

Genes Dev, 1997 Apr 15, 11(8), 1037 - 47
A shared subunit belongs to the eukaryotic core RNA polymerase; Lanzendorfer M et al.; The yeast RNA polymerase I is a multimeric complex composed of 14 distinct subunits, 5 of which are shared by the three forms of nuclear RNA polymerase . The reasons for this structural complexity are still largely unknown . Isolation of an inactive form of RNA Pol I lacking the A43, ABC23, and A14 subunits (RNA Pol I delta) allowed us to investigate the function of the shared subunit ABC23 by in vitro reconstitution experiments . Addition of recombinant ABC23 alone to the RNA Pol I delta reactivated the enzyme to up to 50% of the wild-type enzyme activity . The recombinant subunit was stably and stoichiometrically reassociated within the enzymatic complex . ABC23 was found to be required for the formation of the first phosphodiester bond, but it was not involved in DNA binding by RNA Pol I, as shown by gel retardation and surface plasmon resonance experiments, and did not recycle during transcription . Electron microscopic visualization and electrophoretic analysis of the subunit depleted and reactivated forms of the enzyme indicate that binding of ABC23 caused a major conformational change leading to a transcriptionally competent enzyme . Altogether, our results demonstrate that the ABC23 subunit is required for the structural and functional integrity of RNA Pol I and thus should be considered as part of the core enzyme.

Cancer Res, 1997 Apr 15, 57(8), 1412 - 5
Ku proteins join DNA fragments as shown by atomic force microscopy; Pang D et al.; The binding of the Ku protein to DNA was investigated using the atomic force microscope . Ku was found to bind predominantly to the ends of double-stranded DNA . Experiments with plasmid DNA revealed that Ku does not bind to circular plasmids but does bind to plasmids that have been linearized by treatment with ionizing radiation . The binding of Ku to poly(dG-dC) x poly(dG-dC) polynucleotides and to a 400-bp DNA EcoRI fragment resulted in a shift in the fragment size distribution to include longer fragments, with internally binding Ku . Furthermore, we observed images consistent with fragments joined together by Ku, showing an interaction with two ends of DNA . These observations suggest that Ku may play a role in physically orienting DNA for ligation by binding the ends of adjacent DNA molecules.

Blood, 1997 Apr 15, 89(8), 2975 - 85
TRA1, a novel mRNA highly expressed in leukemogenic mouse monocytic sublines but not in nonleukemogenic sublines; Kasukabe T et al.; Mouse monocytic Mm-A, Mm-P, Mm-S1, and Mm-S2 cells are sublines of mouse monocytic and immortalized Mm-1 cells derived from spontaneously differentiated, mouse myeloblastic M1 cells . Although these subline cells retain their monocytic characteristics in vitro, Mm-A and Mm-P cells are highly leukemogenic to syngeneic SL mice and athymic nude mice, whereas Mm-S1 and Mm-S2 cells are not or are only slightly leukemogenic . To better understand the molecular mechanisms of these levels of leukemogenicity, we investigated putative leukemogenesis-associated genes or oncogenes involved in the maintenance of growth, especially in vivo, by means of differential mRNA display . We isolated a fragment clone (15T01) from Mm-P cells . The mRNA probed with 15T01 was expressed at high levels in leukemogenic Mm-P and Mm-A cells but not in nonleukemogenic Mm-S1 and Mm-S2 cells . The gene corresponding to 15T01, named TRA1, was isolated from an Mm-P cDNA library . The longest open reading frame of the TRA1 clone predicts a peptide containing 204 amino acids with a calculated molecular weight of 23,049 D . The predicted TRA1 protein is cysteine-rich and contains multiple cysteine doublets . A putative normal counterpart gene, named NOR1, was also isolated from a normal mouse kidney cDNA library and sequenced . NOR1 cDNA predicts a peptide containing 234 amino acids . The sequence of 201 amino acids from the C-terminal NOR1 was completely identical to that of TRA1, whereas the remaining N-terminal amino acids (33 amino acids) were longer than that (3 amino acids) of TRA1 and the N-terminus of NOR1 protein contained proline-rich sequence . A similarity search against current nucleotide and protein sequence databases indicated that the NOR1/TRA1 gene(s) is conserved in a wide range of eukaryotes, because apparently homologous genes were identified in Caenorhabditis elegans and Saccharomyces cerevisiae genomes . Northern blotting using TRA1-specific and NOR1-specific probes indicated that TRA1 mRNA is exclusively expressed in leukemogenic but not in nonleukemogenic Mm sublines and normal tissues and also indicated that NOR1 mRNA is expressed in normal tissues, especially in kidney, lung, liver, and bone marrow cells but not in any Mm sublines . After leukemogenic Mm-P cells were induced to differentiate into normal macrophages by sodium butyrate, the normal counterpart, NOR1, was expressed, whereas the TRA1 level decreased . Furthermore, transfection of TRA1 converted nonleukemogenic Mm-S1 cells into leukemogenic cells . These results indicate that the TRA1 gene is associated at least in part with the leukemogenesis of monocytic Mm sublines.

Proc Natl Acad Sci U S A, 1997 Apr 15, 94(8), 3656 - 61
The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase; Huibregtse JM et al.; The E3 ubiquitin-protein ligases play an important role in controlling substrate specificity of the ubiquitin proteolysis system . A biochemical approach was taken to identify substrates of Rsp5, an essential hect (homologous to E6-AP carboxyl terminus) E3 of Saccharomyces cerevisiae . We show here that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II (Rpb1) in vitro . Stable complex formation between Rsp5 and Rpb1 was also detected in yeast cell extracts, and repression of RSP5 expression in vivo led to an elevated steady-state level of Rpb1 . The amino-terminal domain of Rsp5 mediates binding to Rpb1, while the carboxyl-terminal domain of Rpb1, containing the heptapeptide repeats characteristic of polymerase II, is necessary and sufficient for binding to Rsp5 . Fusion of the Rpb1 carboxyl-terminal domain to another protein also causes that protein to be ubiquitinated by Rsp5 . These findings indicate that Rsp5 targets at least a subset of cellular Rpb1 molecules for ubiquitin-dependent degradation and may therefore play a role in regulating polymerase II activities . In addition, the results support a model for hect E3 function in which the amino-terminal domain mediates substrate binding, while the carboxyl-terminal hect domain catalyzes ubiquitination of bound substrates.

Nucleic Acids Res, 1997 Apr 15, 25(8), 1476 - 84
The bifunctional DCOH protein binds to HNF1 independently of its 4-alpha-carbinolamine dehydratase activity; Sourdive DJ et al.; HNF1 is a liver enriched atypical homeoprotein isolated from vertebrates which is involved in the transcriptional activation of liver, kidney, intestine and pancreas specific genes . HNF1 contains an N-terminal dimerisation and a POU-like domain both essential together with the homeodomain for DNA specific recognition . Using the yeast two-hybrid system we searched for proteins interacting with HNF1 . We repeatedly obtained cDNA clones encoding DCOH/4-alpha-carbinolamine dehydratase, an enzyme involved in the oxidation of aromatic amino acids that was shown to bind to and stabilise HNF1 dimers . Using the yeast system, we show that the enzymatic activity of DCOH is not essential for HNF1 binding and that the HNF1 dimerisation domain is sufficient for DCOH binding . Furthermore we demonstrate that both proteins co-localise in co-transfected cells.

J Biol Chem, 1997 Apr 11, 272(15), 10065 - 71
Identification of regions within the four small subunits of human replication factor C required for complex formation and DNA replication; Uhlmann F et al.; Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are processivity factors for eukaryotic DNA polymerases delta and epsilon . RFC binds to a DNA primer end and loads PCNA onto DNA in an ATP-dependent reaction . The five RFC subunits p140, p40, p38, p37, and p36, all of which are required to form the active RFC complex, share regions of high homology including the defined RFC boxes II-VIII . RFC boxes III and V constitute a putative ATP binding site, whereas the function of the other conserved boxes is unknown . To study the individual subunits in the RFC complex and the role of the RFC boxes, deletion mutations were created in all subunits . Sequences close to the C terminus of each of the small subunits are required for formation of the five subunit complex . A N-terminal region of the small subunits, containing the RFC homology box II, plays a critical role in the function of these subunits, deletion of which reduces but does not abolish RFC activity in loading PCNA onto DNA and in supporting an RFC-dependent replication reaction . The N termini of p37 and p40, although highly homologous, are not interchangeable, suggesting unique functions for the individual subunits.

J Biol Chem, 1997 Apr 11, 272(15), 10058 - 64
Deletion analysis of the large subunit p140 in human replication factor C reveals regions required for complex formation and replication activities; Uhlmann F et al.; Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are processivity factors for eukaryotic DNA polymerases delta and epsilon . RFC contains multiple activities, including its ability to recognize and bind to a DNA primer end and load the ring-shaped PCNA onto DNA in an ATP-dependent reaction . PCNA then tethers the polymerase to the template allowing processive DNA chain elongation . Human RFC consists of five distinct subunits (p140, p40, p38, p37, and p36), and RFC activity can be reconstituted from the five cloned gene products . To characterize the role of the large subunit p140 in the function of the RFC complex, deletion mutants were created that defined a region within the p140 C terminus required for complex formation with the four small subunits . Deletion of the p140 N-terminal half, including the DNA ligase homology domain, resulted in the formation of an RFC complex with enhanced activity in replication and PCNA loading . Deletion of additional N-terminal amino acids, including those constituting the RFC homology box II that is conserved among all five RFC subunits, disrupted RFC replication function . DNA primer end recognition and PCNA binding activities, located in the p140 C-terminal half, were unaffected in this mutant, but PCNA loading was abolished.

J Biol Chem, 1997 Apr 11, 272(15), 9907 - 14
5'-flanking sequences in thyroid hormone response element half-sites determine the requirement of retinoid X receptor for receptor-mediated gene expression; Olson DP et al.; Thyroid hormone receptors are ligand-inducible transcription factors that can potentially interact with thyroid hormone response elements as homodimers or heterodimers with the retinoid X receptor . It has generally been felt, however, that the heterodimer is responsible for induction of gene expression . We have demonstrated previously that the optimal thyroid hormone receptor binding sequence is not the consensus hexamer half-site AGGTCA but is an octamer, TAAGGTCA . Based upon these findings, we hypothesize that thyroid hormone response elements composed of optimal half-sites (TAAGGTCA) will bind thyroid hormone receptors readily and activate gene expression independently of the retinoid X receptor . In contrast, response elements composed of suboptimal half-sites (e.g . GCAGGTCA) will require the retinoid X receptor to facilitate thyroid hormone receptor-mediated gene expression . To test this hypothesis, we have reconstituted thyroid hormone receptor-mediated gene expression in yeast . Our studies confirm the hypothesis that the retinoid X receptor is required for gene expression from response elements composed of suboptimal half-sites, whereas thyroid hormone receptors are sufficient to activate gene expression maximally from response elements containing optimal half-sites . Furthermore, coexpression of steroid receptor coactivator-1 is required for ligand-dependent gene activation from single response elements . Surprisingly, however, coexpression of the retinoid X receptor decreases the steroid receptor coactivator-1-dependent thyroid hormone induction . Overall these data demonstrate that the architecture of the thyroid hormone response element dictates the nuclear receptor requirements for gene activation . The studies suggest that different coactivators may be required for gene activation depending upon the response element architecture and the nature of the bound thyroid hormone receptor complex (homo- versus heterodimer).

FEBS Lett, 1997 Apr 7, 406(1-2), 205 - 10
Comparative processing of bovine leukemia virus envelope glycoprotein gp72 by subtilisin/kexin-like mammalian convertases; Zarkik S et al.; Intracellular proteolytic processing of bovine leukemia virus (BLV) envelope glycoprotein precursor (gp72) at the C-terminal end of the RVRR268 / site is an essential step for virus infectivity . Subtilisin/kexin-like convertases cleave proproteins at preferred RX(K/R)R / sites, including those commonly found in viral envelope glycoprotein precursors . We first demonstrated that gp72 is processed into gp51/gp30 in both CV1 cells and the furin-deficient LoVo cells, leading us to compare the ability of mammalian convertases to cleave BLV gp72 in vitro . In contrast to the inability of the neuroendocrine PC1 to cleave gp72, the convertases furin, PACE4, PC5-A and PC5-B, which process constitutively secreted precursors, can effectively cleave gp72 into gp51/gp30 . N-terminal sequence analysis of the convertase-generated gp30 demonstrated that cleavage occurs at the in vivo-utilized RVRR / SPV site . Such furin-, PACE4- and PC5-mediated processing was completely inhibited by the alpha1-antitrypsin variant alpha1-PDX . Mutagenesis of the gp72 cleavage site into RVRG-TPV resulted in complete abrogation of gp72 processing by endogenous CV-1 cells and by convertases in vitro . Since our in vitro data suggest a redundancy in the ability of the convertases to cleave gp72, RT-PCR analysis was used to define the convertases expressed in B-lymphocytes, representing one of the major targets of BLV infection . Our data revealed that only furin and the newly discovered PC7 mRNAs are expressed in Raji, B-Jab and LG2 cell lines.

Biochim Biophys Acta, 1997 Apr 4, 1338(2), 244 - 52
A N(alpha)-acetyltransferase selectively transfers an acetyl group to NH2-terminal methionine residues: purification and partial characterization; Lee FJ et al.; Methionine N(alpha)-acetyltransferase (M-N(alpha)AT), which selectively catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of methionine residues in proteins and peptides, was isolated from Saccharomyces cerevisiae . The enzyme was purified 22000-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, DE-52 cellulose, CM-52 cellulose, Affi-Gel Blue gel and hydroxyapatite . The Mr of the native enzyme was estimated to be 70000 +/- 5000 by gel filtration chromatography . The enzyme has a pI near 8.3 as determined by chromatofocusing on Mono P . The enzyme catalyzed the transfer of an acetyl group to a synthetic peptide mimicking the first 24 residues of yeast proteinase A inhibitor 3 (Met-Asn-Thr...) and 3 of its 19 penultimately substituted analogues ({Asp2}, {Glu2}, and {Gln2}) . Based on the estimated molecular weight and amino-acid sequence, The enzyme is different from two other recently identified methionine N(alpha)-acetyltransferases, NAT2 (Kulkarni, M.S . and Sherman, F . (1994) J . Biol . Chem . 269, 13141-13147) and MAK3 (Tercero, J.C . and Wickner, R.B . (1992) J . Biol . Chem . 267, 20277-20281) . Among these three enzymes, M-N(alpha)AT and NAT2 have similar substrate specificity, however, only purified M-N(alpha)AT, but not recombinant NAT2 gene product, can catalyze the transfer of acetyl group to NH2-terminal methionine residues . The availability of this methionine N(alpha)-acetyltransferase will advance the understanding of protein co-translational processing.

J Mol Biol, 1997 Apr 4, 267(3), 537 - 47
Suppressors of cis-acting splicing-deficient mutations that affect the ribozyme core of a group II intron; Robineau S et al.; Many of the cis-dominant mutations that lead to respiratory deficiency by preventing maturation of specific yeast mitochondrial transcripts are found to affect the ribozyme core of group I and group II introns . We have searched for suppressors of mutations in the ribozyme-encoding sections of a group II intron, the first intron in the COX1 gene of Saccharomyces cerevisiae, which was independently subjected to in vitro site-directed mutagenesis . Three of the original mutants bore multiple mutations, which act synergistically, since for most individual mutations, suppressors could be obtained that ensured at least partial recovery of respiratory competence and splicing . Out of a total of ten suppressor mutations that were identified, three were second-site substitutions that restored postulated base-pairings in the ribozyme core . Remarkably, and as is observed for group I introns, at least half of the cis-dominant mutations in the first two group II introns of the COX1 gene affect sites that have been shown to participate in RNA tertiary interactions . We propose that this bias reflects cooperativity in the formation of ribozyme tertiary but not secondary structure, on the one hand, and the need for synergistic effects in order to generate a respiratory-deficient phenotype in the laboratory on the other . Finally, a novel in vivo splicing product of mutant cells is attributed to bimolecular splicing at high concentrations of defective transcripts.

J Mol Biol, 1997 Apr 4, 267(3), 520 - 36
Multiple tertiary interactions involving domain II of group II self-splicing introns; Costa M et al.; The ribozyme core of group II introns is organized into six domains of secondary structure . Of these, domain II was long thought to be relatively unimportant for group II self-splicing . However, we now demonstrate the existence, in both major subdivisions of the group II family, of essential tertiary interactions involving domain II . theta-theta' is a novel tertiary interaction between the terminal loop of the IC1 stem of domain I and the basal stem of domain II . The theta-theta' interaction appears to stabilize the group II ribozyme core: it is essential for efficient self-splicing at elevated temperatures but, as shown by the use of a bimolecular reaction system, molecules with a defective theta-theta' contact are not affected in catalysis . An interaction, eta-eta', between domains II and VI of subgroup IIB introns was recently reported to mediate a conformational rearrangement between the two steps of the self-splicing reaction . We now show that domains II and VI of subgroup IIA introns also contact each other, although in a somewhat different way . Reinforcement of the eta-eta' interaction of a subgroup IIA intron prevents the use of a specific 2'-hydroxyl group in domain VI to initiate splicing by transesterification at the 5' splice site; the 5' intron-exon junction is hydrolyzed instead . Since disruption of eta-eta' has exactly opposite effects, and promotes reversal of the first transesterification step, it is concluded that formation of eta-eta' mediates a conformational change in subgroup IIA introns as well . Just like the eta-eta' interaction of subgroup IIB introns, the eta-eta' interaction of subgroup IIA introns (and the theta-theta' interaction) involves terminal loops of the GNRA family and their RNA receptors . Therefore, these motifs are used by nature not only to stabilize three-dimensional RNA architectures, but also in situations that require dynamic interactions.

J Biol Chem, 1997 Apr 4, 272(14), 9221 - 6
Identification and functional expression of HAH1, a novel human gene involved in copper homeostasis; Klomp LW et al.; To search for a mammalian homologue of ATX1, a human liver cDNA library was screened and a cDNA clone was isolated, which encodes a protein with 47% amino acid identity to Atx1p including conservation of the MTCXGC copper-binding domain . RNA blot analysis using this cDNA identified an abundant 0.5-kilobase mRNA in all human tissues and cell lines examined . Southern blot analysis using this same clone indicated that the corresponding gene exists as a single copy in the haploid genome, and chromosomal localization by fluorescence in situ hybridization detected this locus at the interface between bands 5q32 and 5q33 . Yeast strains lacking copper/zinc superoxide dismutase (SOD1) are sensitive to redox cycling agents and dioxygen and are auxotrophic for lysine when grown in air, and expression of this human ATX1 homologue (HAH1) in these strains restored growth on lysine-deficient media . Yeast strains lacking ATX1 are deficient in high affinity iron uptake and expression of HAH1 in these strains permits growth on iron-depleted media and results in restoration of copper incorporation into newly synthesized Fet3p . These results identify HAH1 as a novel ubiquitously expressed protein, which may play an essential role in antioxidant defense and copper homeostasis in humans.

J Biol Chem, 1997 Apr 4, 272(14), 9071 - 7
Cell cycle-dependent transcription of CLN1 involves swi4 binding to MCB-like elements; Partridge JF et al.; Two promoter elements have been defined that activate G1/S-specific transcription in Saccharomyces cerevisiae . SCB elements (CACGAAA) are activated by the Swi4-Swi6 complex, and MCB elements (ACGCGTNA) are activated by the Mbp1-Swi6 complex . CLN1 encodes a cyclin which is expressed during this interval, and requires Swi4 and Swi6 for peak transcription, but it has no consensus SCB elements in its promoter . Two SCB-like sequences had been previously noted and suggested to be the functional promoter elements . Our studies indicate that these sequences are unable to activate transcription of a lacZ reporter construct, or to bind Swi4-Swi6 complexes in vitro . However, a cluster of three sequences resembling MCB sequences are active promoter elements, sufficient to confer G1/S-specific transcription to a reporter . These sites are the predominant activation elements in the CLN1 promoter, and despite their resemblance to MCB elements, they bind Swi4-Swi6 complexes in vitro and require Swi4 and Swi6 for their activity in vivo . This indicates that the sequences that promote Swi4/Swi6 binding have not been fully defined, or that there are multiple Swi4- and Swi6-containing complexes with distinct DNA binding specificities . In addition to these novel Swi4/Swi6-binding sites, these studies also show that there must be at least one novel promoter element that can confer G1/S-specific transcription to CLN1, because when all the potential SCB- and MCB-like sequences are eliminated the transcript is still cell cycle regulated.

J Biol Chem, 1997 Apr 4, 272(14), 8905 - 11
The p120-v-Abl protein interacts with E2F-1 and regulates E2F-1 transcriptional activity; Birchenall-Roberts MC et al.; The E2F family of transcription factors regulates cell cycle progression, and deregulated expression of E2F-1 can lead to neoplastic transformation . In myeloid cells, introduction and expression of the Abelson leukemia virus causes growth factor independence . Here, the p120 v-Abl protein activates E2F-1-mediated transcription through a physical interaction with the E2F-1 transcription factor . BCR-Abl and c-Abl also stimulate E2F-1-mediated transcription . Our results suggest a new mechanism by which v-Abl leads to factor-independent myeloid cell proliferation: the activation of E2F-1-mediated transcription.

Oncogene, 1997 Apr 3, 14(13), 1531 - 9
T-cell oncogene rhombotin-2 interacts with retinoblastoma-binding protein 2; Mao S et al.; The LIM domain protein rhombotin-2 (RBTN-2/TTG-2/Lmo2) has distinct functions in erythropoiesis and in T-cell leukemogenesis . Additional functions for RBTN2 are indicated by its expression in non-hematopoietic tissues . These diverse functions of RBTN2 are presumed to be accomplished through physical interaction with different protein partners that bind the LIM domains of RBTN2 . To identify these proteins which may modulate the activity of RBTN2, a human cDNA library was screened using the yeast two-hybrid assay . Using the RBTN2 LIM domain region as 'bait', the retinoblastoma-binding protein 2 (RBP2) was identified as a partner for RBTN2 . The interaction between RBTN2 and RBP2 was confirmed using in vitro binding assays, and by co-immunoprecipitation of the two proteins . Deletion analysis showed the second LIM domain of RBTN2 was necessary and sufficient for binding to the last 69 amino acids of RBP2 . The interaction between RBTN2 and RBP2 had a functional consequence: the combination of RBP2 and RBTN2 gave higher transcription in vitro, than RBTN2 alone . The interaction with RBP2 suggests two additional functions for RBTN2: (i) RBTN2 may directly affect the activity of RBP2, and/or (ii) RBTN2 may indirectly modulate the functions of the retinoblastoma protein by binding to RBP2.

Nature, 1997 Apr 3, 386(6624), 437 - 8
Cutting complexity down to size; Stuart DI et al.; The proteasome is the most complex eukaryotic macromolecular assembly yet seen in fine detail . The structure reveals completely unexpected mechanisms by which the proteasome neatly chops up unwanted proteins for disposal or display to the immune system.

Mol Biol Cell, 1997 Apr, 8(4), 577 - 82
Phosphatidylinositol 3-kinase is not required for recycling of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network; Nakajima Y et al.; Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrolases from the trans-Golgi network to endosomes, then return to the trans-Golgi network for another round of enzyme delivery . Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, interferes with the delivery of newly synthesized lysosomal enzymes to lysosomes . We used two independent assays of mannose 6-phosphate receptor trafficking to determine the precise step that is blocked by wortmannin . Using an assay that monitors resialylation of desialylated cell surface 300-kDa mannose 6-phosphate receptors, we found that receptor endocytosis and transport to the trans-Golgi network were not inhibited by 2 microM wortmannin . In addition, this concentration of drug had no effect on the transport of the mannose 6-phosphate receptor from late endosomes to the trans-Golgi network using a system that reconstitutes this transport process in cell extracts . Under the same conditions, wortmannin significantly inhibited the generation of mature cathepsin D . In addition, the structurally unrelated phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when added to in vitro endosome-trans-Golgi network transport reactions . These experiments demonstrate that the interruption in lysosomal enzyme targeting is most likely due to a wortmannin-sensitive process required for the export of these receptors from the trans-Golgi network, consistent with the established role of phosphatidylinositol 3-kinase in the equivalent transport process in Saccharomyces cerevisiae.

Leukemia, 1997 Apr, 11 Suppl 3, 486 - 8
MafB represses erythroid genes and differentiation through direct interaction with c-Ets-1; Sieweke MH et al.; Using a yeast interaction screen with a DNA-bound Ets-1 protein we have identified MafB as a direct interaction partner . This distant AP-1 relative, which is specifically expressed in myelomonocytic cells, binds to the DNA binding domain of Ets-1 via its basic region/leucine zipper domain . MafB represses Ets-1 transactivation of synthetic promoters containing Ets binding sites in yeast as well as avian cells . Both Ets-1 and Maf family proteins have been implicated in erythroid specific gene expression . Here we show that MafB inhibits Ets-1-mediated transactivation of the transferrin receptor, which is essential for heme synthesis and erythroid differentiation . Consequently, overexpression of MafB in an erythroblast cell line down-regulates the endogenous transferrin receptor gene and inhibits the cells' potential to differentiate into erythrocytes, without affecting cellular proliferation.

Leukemia, 1997 Apr, 11 Suppl 3, 363 - 6
Kip1 degradation via the ubiquitin-proteasome pathway; Tam SW et al.; The cell cycle has been the object of extensive studies for the past years . A complex network of molecular interactions has been identified . In particular, a class of cell cycle inhibitory proteins has been identified but details of the molecular mechanism of their action have yet to be resolved . These inhibitors regulate the progression through G1 and the G1/S transition via the inhibition of the cyclin-dependent kinase (Cdk) activity . The potential function of these negative regulators as tumor suppressors provides new insights into the link between the cell cycle and oncogenesis . Kip1 is a potent inhibitor of Cdks . In quiescent cells Kip1 accumulates without an increase in mRNA or protein synthesis . We demonstrated that cell cycle regulation of Kip1 levels, both in normal and transformed human cells, occurs via the ubiquitin-proteasome pathway . In a crude in vitro system, Kip1 is ubiquitinated and degraded in an ATP dependent manner and inhibition or depletion of the proteasome blocks Kip1 degradation . Human Ubc2 and Ubc3, the homologs of yeast Rad6 and Cdc34 gene products respectively, are specifically involved in the ubiquitination of Kip1 . Compared to proliferating cells, quiescent cells contain a far lower amount of Kip1 ubiquitinating activity . These results represent the first demonstration that the ubiquitin-proteasome pathway plays a role in the regulation of a cell cycle protein in human cells, namely the Cdk inhibitor Kip1 . The specific proteolysis of Kip1 may be involved in the pathway of inactivation of Cdks.

Mech Dev, 1997 Apr, 63(1), 75 - 87
Lethal of scute requires overexpression of daughterless to elicit ectopic neuronal development during embryogenesis in Drosophila; Giebel B et al.; Classical genetics indicates that the achaete-scute gene complex (AS-C) of Drosophila promotes development of neural progenitor cells . To further analyze the function of proneural genes, we have studied the effects of Gal4-mediated expression of lethal of scute, a member of the AS-C, during embryogenesis . Expression of lethal of scute forces progenitor cells of larval internal sensory organs, which are normally committed to this fate independently of the activity of the AS-C, to take on features of external sensory organs . Supernumerary neural cells can be induced ectopically only if daughterless is overexpressed, either alone or together with lethal of scute: cells of the amnioserosa and the hindgut then express neuronal markers . Furthermore, cells of the proctodeal anlage, which normally lack neural competence, acquire the ability to develop as neuroblasts following transplantation into the neuroectoderm . We show here that activated Notch prevents the cells of the neuroectoderm from forming extra neural tissue when they express an excess of proneural proteins . Under the present conditions, lateral inhibition is thus dominant over the activity of proneural genes.

Appl Microbiol Biotechnol, 1997 Apr, 47(4), 364 - 7
Functional roles of protein domains on rice alpha-amylase activity; Terashima M et al.; Characteristics of two rice alpha-amylases Amy1A and Amy3D, and those of two chimeric enzymes Amy1A/3D and Amy3D/1A, engineered from the two isozymes, were compared in the light of the functional roles of protein domains in alpha-amylase . The enzymes that have an Amy1A-type N-terminal domain, Amy1A and Amy1A/3D, showed high activity against soluble starch, while the enzymes that have an Amy3D-type barrel structure, Amy3D and Amy1A/3D, showed high activity in oligosaccharide hydrolysis . Rigidity of protein folding also significantly affected the enzyme activity in both soluble starch and oligosaccharide hydrolysis . Thus, the present work suggests that the structure of the N-terminal domain is important for stability and soluble starch hydrolysis, while the barrel structure that forms the active site significantly affects enzyme activities in oligosaccharide degradation . We have already characterized two rice alpha-amylase isozymes, Amy1A and Amy3D, and a chimeric enzyme engineered from these two isozymes, Amy1A/3D (Terashima et al . 1995, 1996a,b) . In spite of the high homology (70%) of their amino acid sequences, Amy1A and Amy3D showed distinct differences in their enzymatic characteristics . The chimeric enzyme Amy1A/3D, which consists of an Amy1A-type N-terminal domain and an Amy3D-type barrel structure, inherited enzymatic characteristics from the both isozymes . In this work, one other chimeric enzyme, Amy3D/1A, which is the counterpart of Amy1A/3D, has been characterized . The characteristics of these four enzymes are discussed in the light of the functional roles of protein domains.

Curr Biol, 1997 Apr 1, 7(4), R245 - 8
Transposable elements: how non-LTR retrotransposons do it; Finnegan DJ; The source of the enzyme activity responsible for the transposition of retrotransposons of the type that lack terminal repeats has at last been identified: in L1Hs elements, it is encoded by the second open reading frame and is a nuclease related to the apurinic repair endonucleases.

Curr Biol, 1997 Apr 1, 7(4), R235 - 7
Vesicle traffic: get your coat!
Schimmoller F, Itin C, Pfeffer S.
The budding of transport vesicles from the Golgi complex is initiated by activation of the small GTPase ARF; the discovery of enzymes that can convert soluble ARF-GDP to the active, membrane-associated form ARF-GTP will shed light on the mechanism and regulation of the formation of transport vesicles.

Yeast, 1997 Apr, 13(5), 475 - 7
Analysis of 21.7 kb DNA sequence from the left arm of chromosome VII reveals 11 open reading frames: two correspond to new genes; Feuermann M et al.; The DNA sequence of a fragment of 21731 bp (nucleotides 87408 to 109138) located on the left arm of chromosome VII from Saccharomyces cerevisiae S288C has been determined using a random cloning strategy followed by an oligonucleotide-directed sequencing . This fragment contains eight complete genes previously sequenced (CLG1, SKI8, VAM7, YPT32, MIG2, SIP2, SPT16 and CHC1), the 5' part of POX1 and two other complete unidentified open reading frames of more than 100 amino acids.

J Cell Sci, 1997 Apr, 110 ( Pt 8), 991 - 1003
Sec12p requires Rer1p for sorting to coatomer (COPI)-coated vesicles and retrieval to the ER; Boehm J et al.; In Saccharomyces cerevisiae cells lacking the Rer1 protein (Rer1p), the type II transmembrane protein Sec12p fails to be retained in the ER . The transmembrane domain of Sec12p is sufficient to confer Rer1p-dependent ER retention to other membrane proteins . In rer1 mutants a large part of the Sec12-derived proteins can escape to the late Golgi . In contrast, rer3 mutants accumulate Sec12-derived hybrid proteins carrying early Golgi modifications . We found that rer3 mutants harbour unique alleles of the alpha-COP-encoding RET1 gene . ret1 mutants, along with other coatomer mutants, fail to retrieve KKXX-tagged type I transmembrane proteins from the Golgi back to the ER . Surprisingly rer3-11(=ret1-12) mutants do not affect this kind of ER recycling . Pulse-chase experiments using these mutants show that alpha-COP and Rer1p function together in a very early Golgi compartment to initiate the recycling of Sec12p-derived hybrid proteins . Rer1p protein may be directly involved in the retrieval process since it also recycles between the early Golgi and ER in a coatomer (COPI)-dependent manner . Rer1p may act as an adapter coupling the recycling of non-KKXX transmembrane proteins like Sec12p to the coatomer (COPI)-mediated backward traffic.

Trends Biochem Sci, 1997 Apr, 22(4), 128 - 32
Histone acetylation: chromatin in action; Wade PA et al.; Histone acetylation acts as a landmark and determinant for chromatin function . Active roles in the transcription and assembly of chromatin have been discovered for histone acetyltransferases and deacetylases . This review highlights these roles and discusses their significance for the maintenance of cell differentiation.

Trends Biochem Sci, 1997 Apr, 22(4), 110 - 3
Highways for protein delivery to the mitochondria; Lithgow T et al.; Messenger RNA (mRNA) localisation is one of the prime mechanisms to ensure protein localisation in the cytoplasm of polarised embryonic cells, and has been well-studied in the development of Xenopus and Drosophila embryos . But what of other cells? Here, we discuss whether the directed transport of mRNA out of the nucleus, following cytoplasmic highways to a specified organelle in the cytoplasm, might also contribute to the exquisite fidelity of protein targeting observed in all eukaryotic cells.

Glycobiology, 1997 Apr, 7(3), 433 - 43
Isolation and characterization of an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9; Kawar Z et al.; As part of our ongoing efforts to characterize the N-glycosylation pathway of lepidopteran insect cells, we have isolated an alpha 1,2-mannosidase homolog from an Sf9 cDNA library . This cDNA contains an open reading frame which encodes a 670 amino acid protein with a calculated molecular weight of 75,225 Da . This protein has two potential N-glycosylation sites, two consensus calcium binding sequences, and is predicted to be a type II integral membrane protein with a 22 amino acid transmembrane domain (residues 31-52) . The amino acid sequence of this protein is 35-57% identical to Drosophila, human, murine, and yeast alpha 1,2-mannosidases . A transcript of approximately 6 kilobases was detected by Northern blot analysis of Sf9 mRNA . Genomic Southern blots probed with an intron-free fragment of the alpha 1,2-mannosidase gene indicated that there are at least two copies or cross-hybridizing variants of this gene in the Sf9 genome . In vivo expression of the cDNA using a recombinant baculovirus produced a protein that released {3H}mannose from {3H}Man9GlcNAc . This activity required calcium, but not magnesium, and was inhibited by 1-deoxymannojirimycin . These results indicate that Sf9 cells encode and express an alpha 1,2-mannosidase with properties similar to those of other eukaryotic processing alpha 1,2-mannosidases.

Comput Appl Biosci, 1997 Apr, 13(2), 131 - 6
Detection of significant patterns by compression algorithms: the case of approximate tandem repeats in DNA sequences; Rivals E et al.; MOTIVATION: Compression algorithms can be used to analyse genetic sequences . A compression algorithm tests a given property on the sequence and uses it to encode the sequence: if the property is true, it reveals some structure of the sequence which can be described briefly, this yields a description of the sequence which is shorter than the sequence of nucleotides given in extenso . The more a sequence is compressed by the algorithm, the more significant is the property for that sequence . RESULTS: We present a compression algorithm that tests the presence of a particular type of dosDNA (defined ordered sequence-DNA): approximate tandem repeats of small motifs (i.e . of lengths < 4) . This algorithm has been experimented with on four yeast chromosomes . The presence of approximate tandem repeats seems to be a uniform structural property of yeast chromosomes.

Biosci Biotechnol Biochem, 1997 Apr, 61(4), 621 - 4
Non-radioactive adenosine 5'-phosphosulfate sulfotransferase assay by coupling with sulfite reductase and O-acetylserine(thiol)lyase; Ara T et al.; Adenosine 5'-phosphosulfate (APS) sulfotransferase is thought to be an enzyme that transfers the sulfo-group of APS to a carrier compound with a thiol group in the assimilatory sulfate reduction pathway of higher plants . We developed a rapid, non-radioactive assay for APS sulfotransferase . Sulfite released by APS sulfotransferase reaction in the presence of excess dithiothreitol was further converted to cysteine by coupling with yeast sulfite reductase and cabbage O-acetylserine(thiol)lyase . The cysteine thus formed was measured colorimetrically . By this method, 5 to 300 nmol of sulfite could be assessed . When the method was applied to APS sulfotransferase, the enzyme activity was APS-dependent with the partially purified enzyme . We could also detect APS sulfotransferase activity in some higher plants by this method.

Plant Cell, 1997 Apr, 9(4), 571 - 82
The syntaxin homolog AtPEP12p resides on a late post-Golgi compartment in plants; da Silva Conceicao A et al.; Soluble proteins are transported to the plant vacuole through the secretory pathway via membrane-bound vesicles . Targeting of vesicles to appropriate organelles requires several membrane-bound and soluble factors that have been characterized in yeast and mammalian systems . For example, the yeast PEP12 protein is a syntaxin homolog that is involved in protein transport to the yeast vacuole . Previously, we isolated an Arabidopsis thaliana homolog of PEP12 by functional complementation of the yeast pep12 mutant . Antibodies raised against the cytoplasmic portion of AtPEP12 have been prepared and used for intracellular localization of this protein . Biochemical analysis indicates that AtPEP12 does not localize to the endoplasmic reticulum, Golgi apparatus, plasma membrane, or tonoplast in Arabidopsis plants; furthermore, based on biochemical and electron microscopy immunogold labeling analyses, AtPEP12 is likely to be localized to a post-Golgi compartment in the vacuolar pathway.

J Spinal Cord Med, 1997 Apr, 20(2), 200 - 6
Axonal sprouting following incomplete spinal cord injury: an experimental model; Goldstein B et al.; Recovery of function following incomplete spinal cord injury may in part result from growth of new connections by spared descending pathways . It has been difficult to demonstrate such anatomical reorganization with traditional anatomic techniques . This study utilizes an immunocytochemical method to demonstrate axonal growth cones within the lumbar spinal cord in rats recovering from an incomplete midthoracic spinal cord injury . Adult rats underwent subtotal section of the midthoracic cord sparing the left lateral funiculus and a portion of the left ventral funiculus . Light microscope immunocytochemistry was performed on sections of lumbar spinal cord with antibodies to identify sprouting axons . These antibodies were used to determine the distribution of growth cones on both sides of the lumbar spinal cord in experimental and control animals . Growth cones were first observed three days after the spinal cord lesion . Specific labeling, similar in appearance to previous reports of growth cone identification, was apparent within the immediate gray and ventral horns on both sides of the cord . These data support the hypothesis of collateral sprouting distal to the lesion site following incomplete spinal cord injury . It further supports the idea that recovery of function following incomplete spinal cord injury is, in part, mediated by spared descending pathways.

Am J Physiol, 1997 Apr, 272(4 Pt 1), E696 - 711
Measurement and modeling of glucose-6-phosphatase in pancreatic islets; Sweet IR et al.; In the beta-cells of the pancreas, glucose phosphorylation carried out by glucokinase is the rate-controlling step in glycolysis, and the kinetic characteristics of glucokinase govern to a high degree the dose-response relationship between glucose and insulin release . Because glucose-6-phosphatase (G-6-Pase) opposes the action of glucokinase, it may have a regulatory role in the release of insulin in response to glucose if the enzyme is present in the beta-cells . A number of researchers have reported finding high levels of G-6-Pase in islets, but quantitation of its activity remains controversial, mainly because of difficulties in solubilizing a particulate enzyme . Therefore a method developed to measure functional glucose phosphorylation activity in intact brain was applied (Chi, M . M.-Y., M . E . Pusateri, J . G . Carter, B . J . Norris, D . B . McDougal, Jr., and O . H . Lowry . Anal . Biochem . 161: 508-513, 1987), and the rates of accumulation and disappearance of 2-deoxyglucose 6-phosphate (DG-6-P) in freshly harvested islets were determined as a measure of glucose cycling . Islets were incubated in the presence of 30 mM 2-deoxyglucose (DG) for 60 min, and subsequently the incubation medium was replaced with medium containing no DG, but instead high levels of mannoheptulose as a blocker of phosphorylation . The content of DG-6-P in the islets was measured at strategic times during the protocol . As predicted by a mathematical model, DG-6-P accumulated in the presence of DG and decayed after its washout . Both of these results are consistent with islets containing dephosphorylation activity for this substrate . The kinetic curves were fit using a mathematical model, and the maximal G-6-Pase activity was estimated to be 0.13 +/- 0.005 micromol x g(-1) x min(-1) . However, when the physiological effect of this amount of G-6-Pase activity was assessed by use of a model of glycolysis, it was found that the impact on glucose cycling and usage was insignificant . It was concluded that normal islets do contain measurable activity for dephosphorylating glucose 6-phosphate but that this enzymatic reaction does not play a role in glucose metabolism and sensing by the normal beta-cell.

Mol Microbiol, 1997 Apr, 24(1), 203 - 16
Molecular characterization of the Aspergillus nidulans treA gene encoding an acid trehalase required for growth on trehalose; d'Enfert C et al.; Aspergillus nidulans conidiospores contain high levels of the non-reducing disaccharide trehalose . We show that upon induction of conidiospore germination, the trehalose pool is rapidly degraded and a glycerol pool is transiently accumulated . A trehalase with an acidic pH optimum was purified from conidiospores . Characterization of the treA gene encoding this trehalase shows that it is homologous to Saccharomyces cerevisiae vacuolar acid trehalase, the product of the ATH1 gene, and to two related proteins of unknown function identified in Mycobacterium tuberculosis and Mycobacterium leprae . A . nidulans mutants that lack acid trehalase activity were constructed by gene replacement at the treA locus . Analysis of these mutants suggests that the treA gene product is localized in the conidiospore wall, is required for growth on trehalose as a carbon source, and is not involved in the mobilization of the intracellular pool of trehalose . Therefore, it is proposed that a cytoplasmic regulatory trehalase is controlling this latter process.

Mol Microbiol, 1997 Apr, 24(1), 105 - 17
The gene encoding the major proline transporter of Aspergillus nidulans is upregulated during conidiospore germination and in response to proline induction and amino acid starvation; Tazebay UH et al.; In Aspergillus nidulans a highly specific L-proline transporter is encoded by the prnB gene which is tightly linked to all other genes involved in proline catabolism . In mycelia, the expression of the prn structural genes is finely co-regulated in response to proline induction and nitrogen/carbon catabolite repression . In this study we establish that prnB expression is also activated during germination of conidiospores . This activation persists until the development of 6 h-old mycelia and it is independent of proline induction mediated by the pathway-specific prnA gene product . We then show that, in mycelia, prnB transcription is activated in response to proline or histidine starvation . This process has two components: a prnA-dependent and a prnA-independent component . A cis-acting element that conforms to the consensus target of the GCN4/CPC1 transcriptional activators mediating amino acid biosynthesis activation in other fungi is involved in the activation of prnB transcription in response to amino acid starvation . We also show that the stimulation of prnB expression in germinating conidiospores is not due exclusively to transient internal amino acid starvation occurring during the transition from conidiospore to mycelium . This is the first report that an amino acid transporter gene is upregulated during development and in response to amino acid starvation and specific amino acid induction.

Bioessays, 1997 Apr, 19(4), 307 - 15
Cyclins, cyclin-dependent kinases and differentiation; Gao CY et al.; Cyclin-dependent kinases and their regulatory subunits, the cyclins, are known to regulate progression through the cell cycle . Yet these same proteins are often expressed in non-cycling, differentiated cells . This review surveys the available information about cyclins and cyclin-dependent kinases in differentiated cells and explores the possibility that these proteins may have important functions that are independent of cell cycle regulation.

J Cell Sci, 1997 Apr, 110 ( Pt 7), 911 - 25
Defining the essential functional regions of the nucleoporin Nup145p; Emtage JL et al.; Studies of the essential nucleoporin Nup145p have shown that its depletion is coincident with a block in RNA export and that deletion of its amino-terminal domain results in clustering of nuclear pore complexes . To further define the functional domains of Nup145p, we have characterized a panel of nup145 mutants . Deletions from both the amino terminus and the carboxy terminus resulted in temperature sensitive mutants that accumulated polyadenylated RNA in the nucleus at the non-permissive temperature . In addition, these mutants also displayed constitutive clustering of nuclear pore complexes in localized patches of the nuclear envelope . These results suggested that an internal region of Nup145p consisting of amino acids 593-893 is essential for function . Accordingly, when this region was deleted, growth was not supported at any temperature, whereas the region alone was able to complement a null mutation when expressed on a high copy plasmid . Previous studies have suggested that Nup145p is cleaved into two polypeptides of approximately 65 and 80 kDa . Interestingly, our experiments suggest that cleavage occurs in vivo . However, a small internal deletion of 17 amino acid residues that abolished cleavage had no effect on cell growth . Therefore, cleavage is not necessary for Nup145p function . When a sequence harboring the Nup145p cleavage site required for Nup145p cleavage was inserted in a chimeric protein, it was not sufficient for mediating cleavage . Cleavage likely requires a second region from amino acid residues 247-524 in addition to the cleavage site.

Gene, 1997 Apr 1, 188(2), 175 - 81
Construction and characterization of a rad51rad52 double mutant as a host for YAC libraries; Kohno K et al.; RAD52 or RAD51 recombination-deficient yeast strains stabilize otherwise unstable YACs containing ribosomal DNA or the human color vision locus (Kohno et al., 1994) . Thus the RAD52RAD51 pathways(s) are apparently involved in the instability of YACs containing tandem repeat loci, presumably by promoting recombination-based deletion formation . Some other genomic loci are still unstable or unrecoverable in those strains, but we now find that greater stability is observed in a rad51rad52 double mutant strain that we have newly constructed . YACs containing a highly unstable region around DXS49 or centromeric regions throw off a variety of products in single mutants, but are much more stable in the rad51rad52 strain, which could therefore provide a better host for library construction and maintenance.

Mol Med, 1997 Apr, 3(4), 248 - 59
A genetic approach to mapping the p53 binding site in the MDM2 protein; Freedman DA et al.; BACKGROUND: The MDM2 oncoprotein binds to the tumor suppressor p53 and inhibits its anti-oncogenic functions . MATERIALS AND METHODS: To determine the amino acids of MDM2 that are critical for binding to p53, a modified two-hybrid screen was performed in yeast . Site-directed mutagenesis was then performed to identify MDM2 residues important for p53 interaction . Mutant MDM2 proteins were subsequently tested for their ability to bind to p53 in vitro and for their ability to regulate p53-mediated transcription in vivo . RESULTS: The yeast genetic screen yielded two Mdm2 mutations (G58D and C77Y) which disrupted binding to p53 in vitro without altering the conformation of MDM2 as determined with conformation-sensitive monoclonal antibodies . Site-directed mutagenesis yielded mutations of two additional amino acids of MDM2 (D68 and V75) that prevented binding to p53 in vitro . The mutant MDM2 proteins were unable to inhibit p53-dependent transcription in vivo, which is consistent with prior indications that a physical interaction between the two proteins is required for MDM2's inhibition of p53 . Finally, the crystal structure of the MDM2-p53 complex shows that two of the four critical residues identified here contact p53 directly, while the remaining two residues play important structural roles in the MDM2 domain . CONCLUSIONS: MDM2 residues G58, D68, V75, and C77 are critical for MDM2's interaction with the p53 protein . Mutation of these residues to alanine prevents MDM2's interaction with p53 in vitro, and MDM2's regulation of p53's transcriptional activity in vivo.

EMBO J, 1997 Apr 1, 16(7), 1785 - 94
TRF1 is a dimer and bends telomeric DNA; Bianchi A et al.; TRF1 is a mammalian telomeric protein that binds to the duplex array of TTAGGG repeats at chromosome ends . TRF1 has homology to the DNA-binding domain of the Myb family of transcription factors but, unlike most Myb-related proteins, TRF1 carries one rather than multiple Myb-type DNA-binding motifs . Here we show that TRF1 binds DNA as a dimer using a large conserved domain near the N-terminus of the protein for TRF1-TRF1 interactions . Dimerization was observed both in a complex with DNA and in the yeast two-hybrid assay . TRF1 dimers were found to require both Myb repeats for the formation of a stable complex with DNA, indicating a parallel between the DNA-binding mode of TRF1 and other Myb-related proteins . TRF1 was found to have a number of biochemical similarities to Rap1p, a distantly related DNA-binding protein that functions at telomeres in yeast . Rap1p and TRF1 both require two Myb motifs for DNA binding and both factors bind along their cognate telomeric sequences without showing strong cooperative interactions between adjacent proteins . Furthermore, TRF1 was found to bend its telomeric site to an angle of -120 degrees . Since Rap1p similarly distorts telomeric DNA, we propose that DNA bending is important for the function of telomeres in yeast and mammals.

EMBO J, 1997 Apr 1, 16(7), 1721 - 31
Developmental acquisition of enhancer function requires a unique coactivator activity; Majumder S et al.; Enhancers are believed to stimulate promoters by relieving chromatin-mediated repression . However, injection of plasmid-encoded genes into mouse oocytes and embryos revealed that enhancers failed to stimulate promoters prior to formation of a two-cell embryo, even though the promoter was repressed in the maternal nucleus of both oocytes and one-cell embryos . The absence of enhancer function was not due to the absence of a required sequence-specific enhancer activation protein, because enhancer function was not elicited even when these proteins either were provided by an expression vector (GAL4:VP16) or were present as an endogenous transcription factor (TEF-1) and shown to be active in stimulating promoters . Instead, enhancer function in vivo required a unique coactivator activity in addition to enhancer-specific DNA binding proteins and promoter repression . This coactivator activity first appeared during mouse development in two- to four-cell embryos, concurrent with the major onset of zygotic gene expression . Competition between various enhancers was observed in these embryos, but not competition between enhancers and promoters, and competition between enhancers was absent in one-cell embryos . Moreover, enhancer function in oocytes could be partially restored by pre-injecting mRNA from cells in which enhancers were active, the same mRNA did not affect enhancer function in two- to four-cell embryos.

EMBO J, 1997 Apr 1, 16(7), 1710 - 20
Regulation of yAP-1 nuclear localization in response to oxidative stress; Kuge S et al.; The YAP1 gene of Saccharomyces cerevisiae encodes a bZIP-containing transcription factor that is essential for the normal response of cells to oxidative stress . Under stress conditions, the activity of yAP-1 is increased, leading to the induced expression of a number of target genes encoding protective enzymes or molecules . We have examined the mechanism of this activation . Upon imposition of oxidative stress, a small increase in the DNA-binding capacity of yAP-1 occurs . However, the major change is at the level of nuclear localization; upon induction the yAP-1 protein relocalizes from the cytoplasm to the nucleus . This regulated localization is mediated by a cysteine-rich domain (CRD) at the C-terminus, its removal resulting in constitutive nuclear localization and high level activity . Furthermore, the CRD of yAP-1 is sufficient to impose regulated nuclear localization of the GAL4 DNA-binding domain . Amino acid substitutions indicated that three conserved cysteine residues in the CRD are essential for the regulation . We suggest therefore, that these cysteine residues are important in sensing the redox state of the cell and hence regulating yAP-1 activity.

EMBO J, 1997 Apr 1, 16(7), 1686 - 94
harakiri, a novel regulator of cell death, encodes a protein that activates apoptosis and interacts selectively with survival-promoting proteins Bcl-2 and Bcl-X(L); Inohara N et al.; Programmed cell death is essential in organ development and tissue homeostasis and its deregulation is associated with the development of several diseases in mice and humans . The precise mechanisms that control cell death have not been elucidated fully, but it is well established that this form of cellular demise is regulated by a genetic program which is activated in the dying cell . Here we report the identification, cloning and characterization of harakiri, a novel gene that regulates apoptosis . The product of harakiri, Hrk, physically interacts with the death-repressor proteins Bcl-2 and Bcl-X(L), but not with death-promoting homologs, Bax or Bak . Hrk lacks conserved BH1 and BH2 regions and significant homology to Bcl-2 family members or any other protein, except for a stretch of eight amino acids that exhibits high homology with BH3 regions . Expression of Hrk induces cell death which is inhibited by Bcl-2 and Bcl-X(L) . Deletion of 16 amino acids including the conserved BH3 region abolished the ability of Hrk to interact with Bcl-2 and Bcl-X(L) in mammalian cells . Moreover, the killing activity of this mutant form of Hrk (Hrk deltaBH3) was eliminated or dramatically reduced, suggesting that Hrk activates cell death at least in part by interacting with and inhibiting the protection afforded by Bcl-2 and Bcl-X(L) . Because Hrk lacks conserved BH1 and BH2 domains that define Bcl-2 family members, we propose that Hrk and Bik/Nbk, another BH3-containing protein that activates apoptosis, represent a novel class of proteins that regulate apoptosis by interacting selectively with survival-promoting Bcl-2 and Bcl-X(L).

Mol Cell Biol, 1997 Apr, 17(4), 2226 - 34
Ku86 is not required for protection of signal ends or for formation of nonstandard V(D)J recombination products; Han JO et al.; Ku, a heterodimer of 70- and 86-kDa subunits, serves as the DNA binding component of the DNA-dependent protein kinase (DNA-PK) . Cells deficient for the 86-kDa subunit of Ku (Ku86-deficient cells) lack Ku DNA end-binding activity and are severely defective for formation of the standard V(D)J recombination products, i.e., signal and coding joints . It has been widely hypothesized that Ku is required for protection of broken DNA ends generated during V(D)J recombination . Here we report the first analysis of V(D)J recombination intermediates in a Ku-deficient cell line . We find that full-length, ligatable signal ends are abundant in these cells . These data show that Ku86 is not required for the protection or stabilization of signal ends, suggesting that other proteins may perform this function . The presence of high levels of signal ends in Ku-deficient cells prompted us to investigate whether these ends could participate in joining reactions . We show that nonstandard V(D)J recombination products (hybrid joints), which involve joining a signal end to a coding end, form with similar efficiencies in Ku-deficient and wild-type fibroblasts . These data support the surprising conclusion that Ku is not required for some types of V(D)J joining events . We propose a novel RAG-mediated joining mechanism, analogous to disintegration reactions performed by retroviral integrases, to explain how formation of hybrid joints can bypass the requirement for Ku and DNA-PK.

Mol Cell Biol, 1997 Apr, 17(4), 2099 - 106
The Snf1 protein kinase and its activating subunit, Snf4, interact with distinct domains of the Sip1/Sip2/Gal83 component in the kinase complex; Jiang R et al.; The Snf1 protein kinase plays a central role in the response to glucose starvation in the yeast Saccharomyces cerevisiae . Previously, we showed that two-hybrid interaction between Snf1 and its activating subunit, Snf4, is inhibited by high levels of glucose . These findings, together with biochemical evidence that Snf1 and Snf4 remain associated in cells grown in glucose, suggested that another protein (or proteins) anchors Snf1 and Snf4 into a complex . Here, we examine the possibility that a family of proteins, comprising Sip1, Sip2, and Gal83, serves this purpose . We first show that the fraction of cellular Snf4 protein that is complexed with Snf1 is reduced in a sip1delta sip2delta gal83delta triple mutant . We then present evidence that Sip1, Sip2, and Gal83 each interact independently with both Snf1 and Snf4 via distinct domains . A conserved internal region binds to the Snf1 regulatory domain, and the conserved C-terminal ASC domain binds to Snf4 . Interactions were mapped by using the two-hybrid system and were confirmed by in vitro binding studies . These findings indicate that the Sip1/Sip2/Gal83 family anchors Snf1 and Snf4 into a complex . Finally, the interaction of the yeast Sip2 protein with a plant Snf1 homolog suggests that this function is conserved in plants.

Mol Cell Biol, 1997 Apr, 17(4), 1904 - 12
The casein kinase II beta subunit binds to Mos and inhibits Mos activity; Chen M et al.; Mos is a germ cell-specific serine/threonine kinase and is required for Xenopus oocyte maturation . Active Mos stimulates a mitogen-activated protein kinase (MAPK) by directly phosphorylating and activating MAPK kinase (MKK) . We report here that the Xenopus homolog of the beta subunit of casein kinase II (CKII beta) binds to and regulates Mos . The Mos-interacting region of CKII beta was mapped to the C terminus . Mos bound to CKII beta in somatic cells ectopically expressing Mos and CKII beta as well as in unfertilized Xenopus eggs . CKII beta inhibited Mos-mediated MAPK activation in rabbit reticulocyte lysates and repressed MKK activation by v-Mos in a coupled kinase assay . In addition, microinjection of CKII beta mRNA into Xenopus oocytes inhibited progesterone-induced meiotic maturation and MAPK activation, presumably by binding of CKII beta to Mos and thereby inhibiting MAPK activation . Moreover, this inhibitory phenotype could be rescued by another protein that binds to CKII beta, CKII alpha . The ability of ectopic CKII beta to inhibit meiotic maturation and the detection of a complex between endogenous Mos and CKII beta suggest that CKII beta may act as an inhibitor of Mos during oocyte maturation, perhaps setting a threshold beyond which Mos protein must accumulate before it can activate the MAPK pathway.

Mol Cell Biol, 1997 Apr, 17(4), 1881 - 9
DNA-binding specificity of Mcm1: operator mutations that alter DNA-bending and transcriptional activities by a MADS box protein; Acton TB et al.; The yeast Mcm1 protein is a member of the MADS box family of transcriptional regulatory factors, a class of DNA-binding proteins found in such diverse organisms as yeast, plants, flies, and humans . To explore the protein-DNA interactions of Mcm1 in vivo and in vitro, we have introduced an extensive series of base pair substitutions into an Mcm1 operator site and examined their effects on Mcm1-mediated transcriptional regulation and DNA-binding affinity . Our results show that Mcm1 uses a mechanism to contact the DNA that has some significant differences from the one used by the human serum response factor (SRF), a closely related MADS box protein in which the three-dimensional structure has been determined . One major difference is that 5-bromouracil-mediated photo-cross-linking experiments indicate that Mcm1 is in close proximity to functional groups in the major groove at the center of the recognition site whereas the SRF protein did not exhibit this characteristic . A more significant difference is that mutations at a position outside of the conserved CC(A/T)6GG site significantly reduce Mcm1-dependent DNA bending, while these substitutions have no effect on DNA bending by SRF . This result shows that the DNA bending by Mcm1 is sequence dependent and that the base-specific requirements for bending differ between Mcm1 and SRF . Interestingly, although these substitutions have a large effect on DNA bending and transcriptional activation by Mcm1, they have a relatively small effect on the DNA-binding affinity of the protein . This result suggests that the degree of DNA bending is important for transcriptional activation by Mcm1.

Mol Cell Biol, 1997 Apr, 17(4), 1868 - 80
Functional dissection of the B" component of RNA polymerase III transcription factor IIIB: a scaffolding protein with multiple roles in assembly and initiation of transcription; Kumar A et al.; Transcription factor IIIB (TFIIIB), the central transcription factor of Saccharomyces cerevisiae RNA polymerase III, is composed of TATA-binding protein, the TFIIB-related protein Brf, and B" . B", the last component to enter the TFIIIB-DNA complex, confers extremely tight DNA binding on TFIIIB . Terminally and internally deleted B" derivatives were tested for competence to form TFIIIB-DNA complexes by TFIIIC-dependent and -independent pathways on the SUP4 tRNA(Tyr) and U6 snRNA (SNR6) genes, respectively, and for transcription . Selected TFIIIB-TFIIIC-DNA complexes assembled with truncated B" were analyzed by DNase I footprinting, and the surface topography of B" in the TFIIIB-DNA complex was also analyzed by hydroxyl radical protein footprinting . These analyses define functional domains of B" and also reveal roles in start site selection by RNA polymerase III and in clearing TFIIIC from the transcriptional start . Although absolutely required for transcription, B" can be extensively truncated . Core proteins retaining as few as 176 (of 594) amino acids remain competent to transcribe the SNR6 gene in vitro . TFIIIC-dependent assembly on DNA and transcription requires a larger core of B": two domains (I and II) that are required for SNR6 transcription on an either-or basis are simultaneously required for TFIIIC-dependent assembly of DNA complexes and transcription . Domains I and II of B" are buried upon assembly of the TFIIIB-DNA complex, as determined by protein footprinting . The picture of the TFIIIB-DNA complex that emerges is that B" serves as its scaffold and is folded over in the complex so that domains I and II are near one another.

Curr Opin Genet Dev, 1997 Apr, 7(2), 152 - 7
Chromatin proteins involved in the initiation of DNA replication; Rowles A et al.; Eukaryotic DNA replication is regulated at least in part by the assembly of initiation proteins onto origins of replication . The origin recognition complex (ORC) is bound to origins throughout most of the cell cycle . Other initiation proteins, such as Cdc6 and the MCM/P1 proteins, are assembled onto ORC-containing chromatin during G1 to define a prereplicative complex . During S phase, these proteins are displaced from chromatin and their reassembly is inhibited by protein-dependent kinases.

Curr Opin Genet Dev, 1997 Apr, 7(2), 212 - 9
RNA movement between the nucleus and the cytoplasm; Lee MS et al.; The past year has seen significant advances in our understanding of the mechanism of RNA movement between the nucleus and the cytoplasm . The emerging view is that proteins bind to and escort RNAs to their proper subcellular location . The discovery of peptide signals that target nuclear export and the identification of novel protein mediators of RNA export are examples of significant recent discoveries.

Curr Opin Genet Dev, 1997 Apr, 7(2), 182 - 91
Chromatin remodeling and transcription; Tsukiyama T et al.; Recent advances highlight two important chromatin remodeling systems involved in the transcriptional process . One system includes several members of the evolutionarily conserved SWI2/SNF2 family found in distinct multiprotein complexes with ATP-dependent nucleosome destabilizing activity; the other is the enzymatic system that governs histone acetylation and deacetylation . Identification of the catalytic subunits of these opposing histone-modifying activities reveal conserved proteins defined genetically as transcriptional regulators.

Curr Opin Genet Dev, 1997 Apr, 7(2), 281 - 7
Mechanisms of cellular senescence; Smeal T et al.; Aging is a near universal process, yet the molecular mechanisms that underlie cellular senescence have remained elusive . Recent progress in determining the roles of various genetic influences in controlling the rate of cellular aging has made this an exciting time in aging research . Genetic screens designed to isolate long-lived mutants in Saccharomyces cerevisiae and Caenorhabditis elegans have implicated factors involved in transcriptional silencing and the dauer pathway in the control of aging . The gene responsible for Werner's syndrome, a disease with symptoms of premature aging, was isolated and found to be a member of the RecQ subfamily of DNA helicases . The regulation of telomere length and its role in senescence and cellular immortalization has been found to be more complex than expected . In C . elegans, mutations have been isolated in maternal-effect genes that presumably control its biological clocks and can dramatically extend its lifespan . Indeed, aging research within the past year has implicated a variety of mechanisms ranging from the control of gene expression, stress resistance, and DNA metabolism to the overall 'rate of living'.

Photochem Photobiol, 1997 Apr, 65(4), 750 - 8
Purification and characterization of recombinant affinity peptide-tagged oat phytochrome A; Murphy JT et al.; Full-length Avena sativa (oat) phytochrome A (ASPHYA) was expressed in the yeast Saccharomyces cerevisiae and purified to apparent homogeneity . Expression of an ASPHYA cDNA that encoded the full-length photoreceptor with a 15 amino acid 'strep-tag' peptide at its C-terminus produced a single polypeptide with a molecular mass of 124 kDa . This strep-tagged polypeptide (ASPHYA-ST) bound tightly to streptavidin agarose and was selectively eluted using diaminobiotin, with a chromatographic efficiency of 45% . Incubation of ASPHYA-ST with phytochromobilin (P phi B) and the unnatural chromophore precursors, phycocyanobilin (PCB) and phycoerythrobilin (PEB), produced covalent adducts that were similarly affinity purified . Both P phi B and PCB adducts of ASPHYA-ST were photoactive--the P phi B adduct displaying spectrophotometric properties nearly indistinguishable from those of the native photoreceptor, and the PCB adduct exhibiting blue-shifted absorption maxima . Although the PEB adduct of ASPHYA-ST was photochemically inactive, it was intensely fluorescent with an excitation maximum at 576 nm and emission maxima at 586 nm . The superimposability of its absorption and fluorescence excitation spectra established that a single biliprotein species was responsible for fluorescence from the adduct produced when ASPHYA-ST was incubated with PEB . Steric exclusion HPLC also confirmed that ASPHYA-ST and its three bilin adducts were homodimers, as has been established for phytochrome A isolated from natural sources . The ability to express and purify recombinant phytochromes with biochemical properties very similar to those of the native molecule should facilitate detailed structural analysis of this important class of photoreceptors.

Plant Physiol, 1997 Apr, 113(4), 1379 - 84
Divergent fructokinase genes are differentially expressed in tomato; Kanayama Y et al.; Two cDNA clones (Frk1 and Frk2) encoding fructokinase (EC 2.7.1.4) were isolated from tomato (Lycopersicon esculentum) . The Frk2 cDNA encoded a deduced protein of 328 amino acids that was more than 90% identical with a previously characterized potato (Solanum tuberosum) fructokinase . In contrast, the Frk1 cDNA encoded a deduced protein of 347 amino acids that shared only 55% amino acid identity with Frk2 . Both deduced proteins possessed and ATP-binding motif and putative substrate recognition site sequences identified in bacterial fructokinases . The Frk1 cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line, which lacks the ability to phosphorylate glucose and fructose and is unable to grow on glucose or fructose . Mutant cells expressing Frk1 were complemented to grow on fructose but not glucose, indicating that Frk1 phosphorylates fructose but not glucose, and this activity was verified in extracts of transformed yeast . The mRNA corresponding to Frk2 accumulated to high levels in young, developing tomato fruit, whereas the Frk1 mRNA accumulated to higher levels late in fruit development . The results indicate that fructokinase in tomato is encoded by two divergent genes, which exhibit a differential pattern of expression during fruit development.

J Histochem Cytochem, 1997 Apr, 45(4), 551 - 8
Phosphatidylinositol transfer protein in lung: cellular and subcellular localization; Ibrahim AM et al.; Determination of the cellular distribution of phosphatidylinositol transfer protein in rat lung by immunocytochemistry revealed that the protein is more readily observed in the nonciliated bronchial epithelial cells (Clara cells) than in other lung cells . By light microscopy, the phosphatidylinositol transfer protein (PtdIns-TP) was localized to the dome-shaped apical region of Clara cells that were identified by staining with an antibody to Clara cell protein . Further investigation by electron microscopy revealed that the PtdIns-TP accumulated at the limiting membrane surrounding secretory granules and at the apical plasma membrane . This localization is compatible with the proposed roles for PtdIns-TP in formation of vesicles and exocytosis of secretory granules and, when considered in the context of the proposed role of PtdIns-TP in phosphatidylinositide metabolism, suggests that phosphatidylinositides may be involved in the mechanisms regulating Clara cell secretion.

FEBS Lett, 1997 Apr 1, 405(3), 305 - 11
RNA polymerase III interferes with Ty3 integration; Connolly CM et al.; Ty3, a gypsylike retrotransposon of budding yeast, integrates at the transcription initiation site of genes transcribed by RNA polymerase III (pol III) . It was previously shown that integration in vitro requires intact promoter elements and the pol III transcription factors TFIIIB and TFIIIC . In order to test the effect of pol III on integration, increasing amounts of a pol III-containing fraction were added to Ty3 in vitro integration reactions . The pol III-containing fraction was inhibitory to integration . These results are consistent with a model where the Ty3 integration complex and pol III recognize similar features of the stable transcription complex and compete with each other for access to the transcription initiation site.

Genes Dev, 1997 Apr 1, 11(7), 941 - 56
The family of box ACA small nucleolar RNAs is defined by an evolutionarily conserved secondary structure and ubiquitous sequence elements essential for RNA accumulation; Ganot P et al.; Eukaryotic cells contain a large number of small nucleolar RNAs (snoRNAs) . A major family of snoRNAs features a consensus ACA motif positioned 3 nucleotides from the 3' end of the RNA . In this study we have characterized nine novel human ACA snoRNAs (U64-U72) . Structural probing of U64 RNA followed by systematic computer modeling of all known box ACA snoRNAs revealed that this class of snoRNAs is defined by a phylogenetically conserved secondary structure . The ACA snoRNAs fold into two hairpin structures connected by a single-stranded hinge region and followed by a short 3' tail . The hinge region carries an evolutionarily conserved sequence motif, called box H (consensus, AnAnnA) . The H box, probably in concert with the flanking helix structures and the ACA box characterized previously, plays an essential role in the accumulation of human U64 intronic snoRNA . The correct processing of a yeast ACA snoRNA, snR36, in mammalian cells demonstrated that the cis- and trans-acting elements required for processing and accumulation of ACA snoRNAs are evolutionarily conserved . The notion that ACA snoRNAs share a common secondary structure and conserved box elements that likely function as binding sites for common proteins (e.g., GAR1) suggests that these RNAs possess closely related nucleolar functions.

Hum Mol Genet, 1997 Apr, 6(4), 627 - 33
Double-strand breaks may initiate the inversion mutation causing the Hunter syndrome; Lagerstedt K et al.; We have previously shown that patients with the Hunter syndrome frequently have suffered from a recombination event between the IDS gene and its putative pseudogene, IDS-2, resulting in an inversion of the intervening DNA . The inversion, which might be the consequence of an intrachromosomal mispairing, is caused by homologous recombination between sequences located in intron 7 of the IDS gene and sequences located distal of exon 3 in IDS-2 . In order to gain insight into the mechanisms causing the inversion, we have isolated both inversion junctions in six unrelated patients . DNA sequence analysis of the junctions showed that all recombinations have taken place within a 1 kb region where the sequence identity is >98% . An interesting finding was the identification of regions with alternating IDS gene and IDS-2 sequences present at one inversion junction, suggesting that the recombination event has been initiated by a double-strand break in intron 7 of the IDS gene . The results from this study suggest that homologous recombination in man could be explained by mechanisms similar to those described for Saccharomyces cerevisiae . The results also have practical implications for diagnosis of patients with the Hunter syndrome.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 3070 - 5
Target of rapamycin proteins and their kinase activities are required for meiosis; Zheng XF et al.; The phosphatidylinositol kinase-related kinases, including Tor1p, Tor2p, FRAP/RAFT, FRP/ATR, ATM, Mec1p, Rad3, and Tel1p, function in signal transduction pathways involved in cell cycle progression and surveillance . The rapamycin-sensitive kinase activities of Tor1p and Tor2p are required for the nutrient-activated protein translation essential for G1 cell cycle progression in haploid yeast cells . In addition, Tor2p's kinase activity is necessary for its unique rapamycin-insensitive function involved in the assembly of the actin cytoskeleton . In the current study using diploid yeast, we found that the kinase activities of the Tor proteins are also required for two discrete steps during yeast meiosisthe switch between the mitotic and meiotic cell cycles and a later step during meiosis involved in the packaging of resultant haploid cells (spores) into asci . Based on what is known of the mitotic functions of Tor and FRAP proteins, these results likely reflect the requirement for signaling pathways leading to regulated protein translation during meiosis . Mec1p, which is required for meiotic recombination, and the Tor proteins are, therefore, homologous kinases with distinct, yet essential, roles in meiosis.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 2885 - 90
Nucleosome-mediated synergism between transcription factors on the mouse mammary tumor virus promoter; Chavez S et al.; In unstimulated mammalian cells and in Saccharomyces cerevisiae, the mouse mammary tumor virus (MMTV) promoter is silent and organized into positioned nucleosomes, one of which encompasses the binding sites for glucocorticoid receptor (GR) and nuclear factor I (NFI) . Glucocorticoid induction in vivo involves a functional synergism between GR and NFI and simultaneous occupancy of the promoter sites for both proteins that cannot be reproduced on naked DNA . The role of chromatin in the process of induction was investigated by manipulating the nucleosome density in yeast strains carrying a regulated histone H4 gene . Following depletion of nucleosomes, independent transactivation by NFI or by GR, as well as binding of the individual proteins to the MMTV promoter, were enhanced, in agreement with a repressive function of nucleosomes . In contrast, NFI-dependent hormone induction of the promoter and the simultaneous binding of receptor and NFI were compromised by nucleosome depletion . This effect could be partly mediated by a cryptic binding site for the receptor that is functional only in the nucleosomal context . Thus, positioned nucleosomes do not only account for constitutive repression of the MMTV promoter, but also participate in induction by mediating cooperative binding and functional synergism between GR and NFI.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 2864 - 8
Synthesis of functional eukaryotic ribosomal RNAs in trans: development of a novel in vivo rDNA system for dissecting ribosome biogenesis; Liang WQ et al.; Active 18S and 25S ribosomal RNAs were produced in trans in yeast, from plasmids containing RNA polymerase II transcription signals and rDNA fragments with unique hybridization tags . Analyses were carried out in cells with temperature-sensitive RNA polymerase I . Functional rRNAs were derived from separate 18S and 5.8/25S rRNA coding units, however, active 25S rRNA could be produced only by cotranscription with 5.8S rRNA . The results demonstrate that the polycistronic organization of the large rDNA operon is not required for successful processing of rRNA or assembly of functional ribosomes . The split operon system should facilitate future efforts to dissect eukaryotic ribosome biogenesis.

Am J Pathol, 1997 Apr, 150(4), 1165 - 72
Polo-like kinase, a novel marker for cellular proliferation; Yuan J et al.; PLK (polo-like kinase) belongs to a family of serine/threonine kinases and represents the human counterpart of polo in Drosophila melanogaster and of CDC5 in Saccharomyces cerevisiae . It is strongly involved in spindle formation and chromosome segregation during mitosis . We have shown previously that PLK mRNA expression correlates with the mitotic activity of cells and the prognosis of lung cancer patients . In this report, the level of PLK protein was analyzed using immunohistochemical techniques . PLK protein was found expressed in the nuclei of tumor cells from lung and breast cancer as well as in several tumor cell lines . Furthermore, in peripheral lymphocytes treated with phytohemagglutinin, elevated proliferative activity of the cells correlated with the up-regulation of PLK protein expression . In contrast, in U937 and HL-60 cells after induction of differentiation with phorbol ester, PLK immunostaining disappeared under conditions of terminal differentiation . Most of the PLK protein was found in the nucleus of proliferating cells with diffuse but distinct staining also in the cytoplasm . Taken together, high levels of PLK protein are associated with cellular proliferation . Combined with other proliferative and oncogene markers, PLK may be useful for improved prediction of the clinical prognosis of cancer patients and for early cancer diagnosis . Due to its activity late in the cell cycle, it may be a target for cancer chemotherapy.

Curr Biol, 1997 Apr 1, 7(4), 253 - 60
Phosphorylation and activation of B-Myb by cyclin A-Cdk2; Ziebold U et al.; BACKGROUND: Cyclins and their catalytic partners, the cyclin-dependent kinases (Cdks), function as key regulators of the eukaryotic cell cycle . Specific cyclin-Cdk complexes are active at successive stages during the cell cycle and control cell-cycle progression by phosphorylating specific target proteins, most of which have not yet been identified . B-Myb, a conserved member of the Myb oncoprotein family, is a sequence-specific DNA-binding protein expressed in virtually all proliferating mammalian cells . Increasing evidence suggests that B-Myb plays an important role during the late G1 and early S phases of the cell cycle . In this study, we have examined the regulation of B-Myb activity by cyclin-Cdks . RESULTS: We found that the transcriptional transactivation potential of B-Myb was repressed by a regulatory domain located at the carboxyl terminus of the protein . Coexpression of B-Myb and cyclin A relieved this repression by phosphorylation of B-Myb in its carboxy-terminal region . Tryptic phosphopeptide mapping revealed that endogenous B-Myb was phosphorylated in cells undergoing S phase . CONCLUSIONS: This work provides evidence for a link between the Myb oncoprotein family and the cell-cycle machinery . We have shown that the carboxyl terminus of B-Myb acts as a cell-cycle sensor that regulates the transactivation function of B-Myb . Moreover, our studies have identified B-Myb as a target of cyclin A-Cdk2 and have indicated that B-Myb activity is regulated by phosphorylation mediated by cyclin A-Cdk2.

Curr Biol, 1997 Apr 1, 7(4), 222 - 7
Muscles in the Drosophila second thoracic segment are patterned independently of autonomous homeotic gene function; Roy S et al.; BACKGROUND: In Drosophila, segment-specific muscle pattern is thought to be determined by the autonomous function of homeotic selector genes in the mesoderm in combination with inductive cues from the developing epidermis and nervous system . Here, we have examined the roles of homeotic genes in the patterning of the somatic muscles of the thoracic segments of Drosophila . RESULTS: We determined the expression patterns of homeoproteins in the mesoderm of the thoracic segments during embryonic and adult development . We found that, unlike the mesoderm of the first and third thoracic segments which express Sex combs reduced and Antennapedia (Antp), respectively, the mesoderm of the second thoracic segment does not express any known homeotic selector gene of the Antp or bithorax complex . In animals homozygous for Antp null mutations, the muscles of the second thoracic segment were affected in the embryo, probably as an indirect consequence of its requirement in the ectoderm . Animals that specifically lacked Antp function in the mesoderm, but expressed the gene in the epidermis, developed with a normal muscle pattern in the second thoracic segment . Furthermore, specific ectopic expression of Antp and other homeotic selector genes in the mesoderm of the second thoracic segment respecified its muscle pattern, indicating that these genes are not required autonomously during muscle development in this segment . Finally, we showed that Antp continues to be expressed in the mesoderm of the homeotically transformed third thoracic segment in the 'four-winged fly', and suggest that this is a likely reason for the failure of flight muscle development in the transformed segment . CONCLUSIONS: We present a model for muscle development in the second thoracic segment whereby mesodermal properties are specified entirely by induction, in contrast to muscle development in other segments, where autonomous function for homeotic selector genes is also required.

Genetics, 1997 Apr, 145(4), 911 - 22
Increased length of long terminal repeats inhibits Ty1 transposition and leads to the formation of tandem multimers; Lauermann V et al.; The Ty1 retrotransposon of Saccharomyces cerevisiae is bounded by long-terminal repeats (LTRs) . We have constructed a variety of Ty1 elements in which the LTR length has been increased from the normal length of 334 bp to > 2 kb . Although small insertions in the LTR have minimal effects on transposition frequency, larger insertions dramatically reduce it . Nevertheless, elements with long LTRs are incorporated into the genome at a low frequency . Most of these rare insertion events represent Ty1 tandem (head to tail) multimers.

RNA, 1997 Apr, 3(4), 382 - 91
A novel protein shared by RNase MRP and RNase P; Chu S et al.; We have isolated suppressors of the temperature-sensitive rRNA processing mutation rrp2-2 in Saccharomyces cerevisiae . A class of extragenic suppressors was mapped to the YBR257w reading frame in the right arm of Chromosome II . Characterization of this gene, renamed POP4, shows that the gene product is necessary both for normal 5.8S rRNA processing and for processing of tRNA . Immunoprecipitation studies indicate that Pop4p is associated with both RNase MRP and RNase P . The protein is also required for accumulation of RNA from each of the two ribonucleoprotein particles.

J Virol, 1997 Apr, 71(4), 3114 - 9
Characterization of the interaction between the baculovirus replication factors LEF-1 and LEF-2; Evans JT et al.; The Autographa californica multinucleocapsid nuclear polyhedrosis virus has six genes required and three genes stimulatory for transient DNA replication . We demonstrate that the products of two of these genes, LEF-1 and LEF-2, interact in both two-hybrid assays using Saccharomyces cerevisiae and glutathione S-transferase fusion affinity assays . Using yeast-two-hybrid assays, we mapped the interaction domain of LEF-2 to amino acids between positions 20 and 60 . Extensive deletion analyses of LEF-1 failed to reveal a delimited interaction domain, suggesting that there may be essential secondary structural elements that are inactivated by these deletions . All clones expressing LEF-1 and LEF-2 that were unable to interact also failed to support significant levels of transient DNA replication, suggesting that this interaction is required for DNA replication . Sequence analysis of LEF-1 revealed a primase-like motif, WVVDAD . When this motif was mutated to WVVQAD, LEF-1 no longer supported transient DNA replication.

Yeast, 1997 Mar 30, 13(4), 299 - 304
Serine palmitoyltransferase (scs1/lcb2) mutants have elevated copy number of the L-A dsRNA virus; Garnepudi VR et al.; The microsomal fraction isolated form serine palmitoyltransferase (lcb2/scs1) mutants is enriched in a 90 kDa protein . The protein was identified as the major coat (Gag) protein of the L-A dsRNA virus particles by partial sequencing and by its interaction with anti-Gag antibodies . The total amount of Gag in whole-cell lysates of scs1/lcb2 mutant cells is greater than in wild-type lysates indicating that the enrichment of the protein in the microsomal fraction of scs1/lcb2 mutant cells may result from increased copy number of the L-A dsRNA virus . This is supported by the findings that the mutants also have increased levels of L-A dsRNA . Altered sphingolipid synthesis in the scs1 mutant cells appears to increase the copy number of the L-A viral particles.

J Biol Chem, 1997 Mar 28, 272(13), 8731 - 8
Topological analysis of the role of homology in Flp-mediated recombination; Azam N et al.; Recombination by the Flp recombinase of Saccharomyces cerevisiae is known to be inhibited by heterology of the overlap regions of the two recombining DNA targets (FRT sites) . We have used topological analysis to show that Flp can promote two rounds of intramolecular recombination between heterologous FRT sites contained within the same supercoiled plasmid . The products are in parental nonrecombinant configuration . Thus, heterology may appear to "block" recombination by rendering the heteroduplex recombinant products unstable, thus favoring a second round of recombination to homoduplex (but parental) products . Hence, homology in the core region is not a requirement for the recombination reaction by Flp but for the formation of recombinant products.

Biochem Biophys Res Commun, 1997 Mar 27, 232(3), 590 - 4
The interaction between human cytomegalovirus immediate-early gene 2 (IE2) protein and heterogeneous ribonucleoprotein A1; Wang YF et al.; Human cytomegalovirus (HCMV) is an ubiquitous pathogen which causes significant illness in immunocompromised individuals . The immediate-early gene 2 (IE2) protein of HCMV plays an important role in the regulation of virus replication . Previous studies have shown that the IE2 protein is able to interact with several cellular proteins, but many of the IE2 interacting partners remain unidentified . By utilizing the yeast two-hybrid system, the heterogeneous ribonucleoprotein A1 (hnRNP A1) was identified as an IE2 interacting protein . The interaction was confirmed via the in vitro binding of bacterial expressed glutathione S-transferase (GST) IE2 fusion protein with the in vitro translated hnRNP A1 . The mutational analysis showed that both the N-terminal half (1-290 residues) and C-terminal half (291-579 residues) of IE2 protein can interact with the hnRNP A1, indicating that more than one region of IE2 protein are involved in the binding with hnRNP A1.

Nature, 1997 Mar 27, 386(6623), 417 - 20
Trans-activation of group II intron splicing by nuclear U5 snRNA; Hetzer M et al.; Similarities between RNA splicing during autocatalytic excision of group II introns and pre-mRNA processing led to the hypothesis that group II introns might be the evolutionary predecessors of spliceosomal small nuclear RNAs . The ID3 subdomain stem-loop structure of group II introns, the proposed analogue of the spliceosomal U5 snRNA, is thought to be essential for 5' splice site recognition and anchoring of the free 5' exon . Using the group II intron bI1 we have analysed the role of ID3 in splicing . In the absence of ID3 the 5' splice site was recognized accurately and efficiently, but exon anchoring was greatly reduced . This step was restored in the presence of RNA fragments consisting of either the terminal stem-loop structure of ID3 or spliceosomal U5 snRNA . This suggests that the predominant role of both RNAs is to anchor the 5' exon during exon ligation . Furthermore, as U5 complements for the loss of ID3, a similar network of structural RNAs may form the catalytic core of both group II introns and spliceosomes.

Science, 1997 Mar 21, 275(5307), 1796 - 800
Modulation of Ras and a-factor function by carboxyl-terminal proteolysis; Boyartchuk VL et al.; Prenylated proteins contain a covalently linked cholesterol intermediate near their carboxyl-termini . Maturation of most prenylated proteins involves proteolytic removal of the last three amino acids . Two genes in Saccharomyces cerevisiae, RCE1 and AFC1, were identified that appear to be responsible for this processing . The Afc1 protein is a zinc protease that participates in the processing of yeast a-factor mating pheromone . The Rce1 protein contributes to the processing of both Ras protein and a-factor . Deletion of both AFC1 and RCE1 resulted in the loss of proteolytic processing of prenylated proteins . Disruption of RCE1 led to defects in Ras localization and signaling and suppressed the activated phenotype associated with the allele RAS2val19.

Nature, 1997 Mar 20, 386(6622), 279 - 84
GRIP: a synaptic PDZ domain-containing protein that interacts with AMPA receptors; Dong H et al.; AMPA glutamate receptors mediate the majority of rapid excitatory synaptic transmission in the central nervous system and play a role in the synaptic plasticity underlying learning and memory . AMPA receptors are heteromeric complexes of four homologous subunits (GluR1-4) that differentially combine to form a variety of AMPA receptor subtypes . These subunits are thought to have a large extracellular amino-terminal domain, three transmembrane domains and an intracellular carboxy-terminal domain . AMPA receptors are localized at excitatory synapses and are not found on adjacent inhibitory synapses enriched in GABA(A) receptors . The targeting of neurotransmitter receptors, such as AMPA receptors, and ion channels to synapses is essential for efficient transmission . A protein motif called a PDZ domain is important in the targeting of a variety of membrane proteins to cell-cell junctions including synapses . Here we identify a synaptic PDZ domain-containing protein GRIP (glutamate receptor interacting protein) that specifically interacts with the C termini of AMPA receptors . GRIP is a new member of the PDZ domain-containing protein family which has seven PDZ domains and no catalytic domain . GRIP appears to serve as an adapter protein that links AMPA receptors to other proteins and may be critical for the clustering of AMPA receptors at excitatory synapses in the brain.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2362 - 7
Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase; Townsley FM et al.; Destruction of mitotic cyclins by ubiquitin-dependent proteolysis is required for cells to complete mitosis and enter interphase of the next cell cycle . In clam eggs, this process is catalyzed by a cyclin-selective ubiquitin carrier protein, E2-C, and the cyclosome/anaphase promoting complex (APC), a 20S particle containing cyclin-selective ubiquitin ligase activity . Here we report cloning a human homolog of E2-C, UbcH10, which shares 61% amino acid identity with clam E2-C and can substitute for clam E2-C in vitro . Dominant-negative clam E2-C and human UbcH10 proteins, created by altering the catalytic cysteine to serine, inhibit the in vitro ubiquitination and destruction of cyclin B in clam oocyte extracts . When transfected into mammalian cells, mutant UbcH10 inhibits the destruction of both cyclin A and B, arrests cells in M phase, and inhibits the onset of anaphase, presumably by blocking the ubiquitin-dependent proteolysis of proteins responsible for sister chromatid separation . Thus, E2-C/UbcH10-mediated ubiquitination is involved in both cdc2 inactivation and sister chromatid separation, processes that are normally coordinated during exit from mitosis.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2295 - 300
Stat5 is a physiological substrate of the insulin receptor; Chen J et al.; Using the cytoplasmic domain of the insulin receptor (IR) in a yeast two-hybrid screen, we identified a cDNA clone encoding the C-terminal 308 amino acids of human Stat5b (Stat5b-Ct) . Stat5b-Ct is tyrosine phosphorylated by purified IR kinase domain in vitro . Insulin stimulates tyrosine phosphorylation of overexpressed Stat5b-Ct and endogenous Stat5 in cells overexpressing IR . Stat5 may be a direct target of the IR and, as a member of the Stat family of transcription factors, may play a role in the regulation of gene transcription by insulin . In support of this hypothesis, perfusion of mouse liver with insulin promotes rapid tyrosine phosphorylation of Stat5 and activation of Stat5 DNA binding . Moreover, refeeding of fasted mice leads to rapid tyrosine phosphorylation and stimulation of enhanced DNA-binding activity of Stat5 extracted from liver, skeletal muscle, and adipose tissues . Taken together, our data strongly suggest that IR interacts with and phosphorylates Stat5 in vitro and in tissues physiologically sensitive to insulin.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2261 - 5
Poly(ADP-ribose) polymerase enhances activator-dependent transcription in vitro; Meisterernst M et al.; Mammalian cells contain activities that amplify the effects of activators on class II gene transcription in vitro . The molecular identity of several of these cofactor activities is still unknown . Here we identify poly(ADP-ribose) polymerase (PARP) as one functional component of the positive cofactor 1 activity . PARP enhances transcription by acting during preinitiation complex formation, but at a step after binding of transcription factor IID . This transcriptional activation requires the amino-terminal DNA-binding domain, but not the carboxyl-terminal catalytic region . In purified systems, coactivator function requires a large molar excess of PARP over the number of templates, as reported for other DNA-binding cofactors such as topoisomerase I . PARP effects on supercoiled templates are DNA concentration-dependent and do not depend on damaged DNA . The PARP coactivator function is suppressed by NAD+, probably as a result of auto-ADP-ribosylation . These observations provide another example of the potentiation of trancription by certain DNA-binding cofactors and may point to interactions of PARP with RNA polymerase II-associated factors in special situations.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2233 - 7
Coordination dynamics of biological zinc "clusters" in metallothioneins and in the DNA-binding domain of the transcription factor Gal4; Maret W et al.; The almost universal appreciation for the importance of zinc in metabolism has been offset by the considerable uncertainty regarding the proteins that store and distribute cellular zinc . We propose that some zinc proteins with so-called zinc cluster motifs have a central role in zinc distribution, since they exhibit the rather exquisite properties of binding zinc tightly while remaining remarkably reactive as zinc donors . We have used zinc isotope exchange both to probe the coordination dynamics of zinc clusters in metallothionein, the small protein that has the highest known zinc content, and to investigate the potential function of zinc clusters in cellular zinc distribution . When mixed and incubated, metallothionein isoproteins-1 and -2 rapidly exchange zinc, as demonstrated by fast chromatographic separation and radiometric analysis . Exchange kinetics exhibit two distinct phases (k(fast) approximately 5000 min(-1) x M(-1); k(slow) approximately 200 min(-1) x M(-1), pH 8.6, 25 degrees C) that are thought to reflect exchange between the three-zinc clusters and between the four-zinc clusters, respectively . Moreover, we have observed and examined zinc exchange between metallothionein-2 and the Gal4 protein (k approximately 800 min(-1) x M(-1), pH 8.0, 25 degrees C), which is a prototype of transcription factors with a two-zinc cluster . This reaction constitutes the first experimental example of intermolecular zinc exchange between heterologous proteins . Such kinetic reactivity distinguishes zinc in biological clusters from zinc in the coordination environment of zinc enzymes, where the metal does not exchange over several days with free zinc in solution . The molecular organization of these clusters allows zinc exchange to proceed through a ligand exchange mechanism, involving molecular contact between the reactants.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2156 - 61
The von Hippel-Lindau tumor-suppressor gene product forms a stable complex with human CUL-2, a member of the Cdc53 family of proteins; Pause A et al.; The inactivation of the von Hippel-Lindau (VHL) gene predisposes affected individuals to VHL syndrome and is an early genetic event associated with sporadic renal cell carcinoma and CNS hemangioblastomas . The VHL protein (pVHL) has been shown to form a stable complex with elongin B and elongin C, two factors that stabilize and activate the transcription elongation factor elongin A . Here, Hs-CUL-2, a member of the recently identified multigene family, the cullins, is shown to specifically associate with the trimeric pVHL-elongin B-C (VBC) complex in vitro and in vivo . Nearly 70% of naturally occurring cancer-predisposing mutations of VHL disrupt this interaction . The pVHL-Hs-CUL-2 association is strictly dependent on the integrity of the trimeric VBC complex . Immunofluorescence studies show Hs-CUL-2 to be a cytosolic protein that can be translocated to the nucleus by pVHL . Recently it has been shown that a yeast Hs-CUL-2 homolog, Cdc53, is part of a ubiquitin protein ligase complex that targets cell cycle proteins for degradation by the ubiquitin proteolytic pathway . In Caenorhabditis elegans, a null mutation of another Hs-cul-2 homolog, Ce-cul-1, results in hyperplasia in all tissues and is required for cell cycle exit . Hence, Hs-cul-2 may be required for VHL function and, therefore, may be a candidate human tumor-suppressor gene.

Gene, 1997 Mar 18, 187(2), 239 - 46
Identification of Cdc45p, an essential factor required for DNA replication; Hardy CF; CDC45 is an essential gene required for initiation of DNA replication in the budding yeast Saccharomyces cerevisiae . CDC45 interacts genetically with CDC46 and CDC47, both members of the MCM family of genes which have been implicated in the licensing of DNA replication . In this report, the isolation of CDC45 is described . The complementing gene is linked to an essential open reading frame on chromosome XII . CDC45 was found to be cell cycle regulated and steady-state mRNA levels are G1/S-specific . CDC45 encodes a protein structurally related to Tsd2p, a protein required for DNA replication in Ustilago maydis . CDC45 also interacts genetically with ORC2, the gene encoding the second subunit of the origin recognition complex, ORC, and MCM3, another member of the MCM family . The cdc45-1 mutant has a plasmid maintenance defect which is rescued by the addition of multiple potential origins to the plasmid.

Gene, 1997 Mar 18, 187(2), 165 - 70
Isolation and characterization of cDNA encoding mouse RNA polymerase II subunit RPB14; Nishi Y et al.; By means of the yeast two-hybrid system using the 40-kDa subunit of mouse RNA polymerase I, mRPA40, as the bait, we isolated a mouse cDNA which encoded a protein with significant homology in amino acid sequence to the 12.5-kDa subunit of Saccharomyces cerevisiae RNA polymerase II, B12.5 (RPB11) . Specific antibody raised against the recombinant protein that was derived from the cDNA reacted with a 14-kDa polypeptide in highly purified mammalian RNA polymerase II and did not react with any subunit of RNA polymerase I or III . Moreover, the antibody co-immunoprecipitated the largest subunit of mouse RNA polymerase II . These results provide biochemical evidence that the cDNA isolated, named mRPB14, encodes a specific subunit of RNA polymerase II, and indicate that the subunit organization of the enzyme is conserved between yeast and mouse . A possible role of the alpha-motif {Dequard-Chablat, M., Riva, M., Carles, C . and Sentenac, A., J . Biol . Chem . 266 (1991) 15300-15307} in the protein-protein interaction between mRPA40 and mRPB14 is also discussed.

EMBO J, 1997 Mar 17, 16(6), 1413 - 26
I kappa B epsilon, a novel member of the I kappa B family, controls RelA and cRel NF-kappa B activity; Whiteside ST et al.; We have isolated a human cDNA which encodes a novel I kappa B family member using a yeast two-hybrid screen for proteins able to interact with the p52 subunit of the transcription factor NF-kappa B . The protein is found in many cell types and its expression is up-regulated following NF-kappa B activation and during myelopoiesis . Consistent with its proposed role as an I kappa B molecule, I kappa B-epsilon is able to inhibit NF-kappa B-directed transactivation via cytoplasmic retention of rel proteins . I kappa B-epsilon translation initiates from an internal ATG codon to give rise to a protein of 45 kDa, which exists as multiple phosphorylated isoforms in resting cells . Unlike the other inhibitors, it is found almost exclusively in complexes containing RelA and/or cRel . Upon activation, I kappa B-epsilon protein is degraded with slow kinetics by a proteasome-dependent mechanism . Similarly to I kappa B-alpha and I kappa B, I kappa B-epsilon contains multiple ankyrin repeats and two conserved serines which are necessary for signal-induced degradation of the molecule . A unique lysine residue located N-terminal of the serines appears to be not strictly required for degradation . Unlike I kappa B- alpha and I kappa B-beta, I kappa B-epsilon does not contain a C-terminal PEST-like sequence . I kappa B-epsilon would, therefore, appear to regulate a late, transient activation of a subset of genes, regulated by RelA/cRel NF-kappa B complexes, distinct from those regulated by other I kappa B proteins.

EMBO J, 1997 Mar 17, 16(6), 1279 - 90
NIK is a new Ste20-related kinase that binds NCK and MEKK1 and activates the SAPK/JNK cascade via a conserved regulatory domain; Su YC et al.; Nck, an adaptor protein composed of one SH2 and three SH3 domains, is a common target for a variety of cell surface receptors . We have identified a novel mammalian serine/threonine kinase that interacts with the SH3 domains of Nck, termed Nck Interacting Kinase (NIK) . This kinase is most homologous to the Sterile 20 (Ste20) family of protein kinases . Of the members of this family, GCK and MSST1 are most similar to NIK in that they bind neither Cdc42 nor Rac and contain an N-terminal kinase domain with a putative C-terminal regulatory domain . Transient overexpression of NIK specifically activates the stress-activated protein kinase (SAPK) pathway . Both the kinase domain and C-terminal regulatory region of NIK are required for full activation of SAPK . NIK likely functions upstream of MEKK1 to activate this pathway; a dominant-negative MEK kinase 1 (MEKK1) blocks activation of SAPK by NIK . MEKK1 and NIK also associate in cells and this interaction is mediated by regulatory domains on both proteins . Two other members of this kinase family, GCK and HPK1, contain C-terminal regulatory domains with homology to that of NIK . These findings indicate that the C-terminal domain of these proteins encodes a new protein domain family and suggests that this domain couples these kinases to the SAPK pathway, possibly by interacting with MEKK1 or related kinases.

EMBO J, 1997 Mar 17, 16(6), 1164 - 72
A specific screen for oligosaccharyltransferase mutations identifies the 9 kDa OST5 protein required for optimal activity in vivo and in vitro; Reiss G et al.; The central reaction in the process of N-linked protein glycosylation in eukaryotic cells, the transfer of the oligosaccharide Glc(3)Man(9)GlcNAc(2) from the lipid dolicholpyrophosphate to selected asparagine residues, is catalyzed by the oligosaccharyltransferase (OTase) . This enzyme consists of multiple subunits; however, purification of the complex has revealed different results with respect to its protein composition . To determine how many different loci are required for OTase activity in vivo, we performed a novel, specific screen for mutants with altered OTase activity . Based on the synthetic lethal phenotype of OTase mutants in combination with a deficiency of dolicholphosphoglucose biosynthesis which results in non-glucosylated lipid-linked oligosaccharide, we identified seven complementation groups with decreased OTase activity . Beside the known OTase loci, STT3, OST1, WBP1, OST3, SWP1 and OST2, a novel locus, OST5, was identified . OST5 is an intron-containing gene encoding a putative membrane protein of 9.5 kDa present in highly purified OTase preparations . OST5 protein is not essential for growth but its depletion results in a reduced OTase activity . Suppression of an ost1 mutation by overexpression of OST5 indicates that this small membrane protein directly interacts with other OTase components, most likely with Ost1p . A strong genetic interaction with a stt3 mutation implies a role in complex assembly.

Eur J Biochem, 1997 Mar 15, 244(3), 852 - 7
Diffusion-dependent kinetic properties of glyoxalase I and estimates of the steady-state concentrations of glyoxalase-pathway intermediates in glycolyzing erythrocytes; Shih MJ et al.; The diffusion-dependent kinetic properties of the yeast glyoxalase I reaction have been measured by means of viscosometric methods . For the glyoxalase-I-catalyzed isomerization of glutathione (GSH)-methylglyoxal thiohemiacetal to S-D-lactoylglutathione, the k(cat)/Km (3.5 x 10(6) M(-1) s(-1), pH 7, 25 degrees C) undergoes a progressive decrease in magnitude with increasing solution viscosity, using sucrose as a viscogenic agent . The viscosity effect is unlikely to be due to a sucrose-induced change in the intrinsic kinetic properties of the enzyme, as the magnitude of k(cat)/Km for the slow substrate GSH-t-butylglyoxal thiohemiacetal (3.5 x 10(3) M(-1) s(-1), pH 7, 25 degrees C) is independent of solution viscosity . Quantitative treatment of the data by means of the Stokes-Einstein diffusion law suggests that catalysis will be about 50% diffusion limited under conditions where {substrate} << Km; the encounter complex between enzyme and substrate partitions nearly equally between product formation and dissociation to form free enzyme and substrate . In a related study, the steady-state concentrations of glyoxalase-pathway intermediates in glycolyzing human erythrocytes are estimated to be in the nanomolar concentration range, on the basis of published values for the activities of glyoxalase I and glyoxalase II in lysed erythrocytes and the steady-state rate of formation of D-lactate in intact erythrocytes . This is consistent with a model of the glyoxalase pathway in which the enzyme-catalyzed steps are significantly diffusion limited under physiological conditions.

Yeast, 1997 Mar 15, 13(3), 233 - 40
Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA; Chun KT et al.; Although the entire DNA sequence of the yeast genome has been determined, the functions of nearly a third of the identified genes are unknown . Recently, we described a collection of mutants, each with a transposon-tagged disruption in an essential gene in Saccharomyces cerevisiae . Identification of these essential genes and characterization of their mutant phenotypes should help assign functions to these thousands of novel genes, and since each mutation in our collection is physically marked by the uniform, unique DNA sequence of the transposable element, it should be possible to use the polymerase chain reaction (PCR) to amplify the DNA adjacent to the transposon . However, existing PCR methods include steps that make their use on a large scale cumbersome . In this report, we describe a semi-random, two-step PCR protocol, ST-PCR . This method is simpler and more specific than current methods, requiring only genomic DNA and two pairs of PCR primers, and involving two successive PCR reactions . Using this method, we have rapidly and easily identified the essential genes identified by several of our mutants.

Genes Dev, 1997 Mar 15, 11(6), 799 - 811
The ROOT HAIR DEFECTIVE3 gene encodes an evolutionarily conserved protein with GTP-binding motifs and is required for regulated cell enlargement in Arabidopsis; Wang H et al.; In plants, morphogenesis is largely determined by the orientation and extent of cell enlargement . To define the molecular mechanisms regulating plant cell enlargement, we have conducted a molecular genetic analysis of the ROOT HAIR DEFECTIVE3 (RHD3) gene of Arabidopsis thaliana . Mutations affecting the RHD3 gene were found to alter cell size, but not cell number, in tissues throughout the plant . Genetic and physiological analyses suggest that the RHD3 gene is not required for proper cell type specification, and it is likely to act downstream of the hormones auxin and ethylene . The RHD3 gene was cloned by a T-DNA tagging method and confirmed by the molecular complementation of the rhd3 mutant phenotype and by the analyses of six rhd3 mutant alleles . Consistent with the global effects of the rhd3 mutations, the RHD3 gene is expressed in all major plant organs . The deduced RHD3 product is a novel 89-kD polypeptide with putative GTP-binding motifs near the amino terminus . RHD3-like genes were identified from a protozoan (Entamoeba histolytica), a fungus (Saccharomyces cerevisiae), and another plant species (Oryza sativa), with the sequence identity including the putative GTP-binding motifs . These results imply that the RHD3 protein is a member of a new class of GTP-binding proteins that is widespread in eukaryotes and required for regulated cell enlargement.

Genomics, 1997 Mar 15, 40(3), 493 - 6
Cloning, expression, and chromosomal assignment of the human mitochondrial intermediate peptidase gene (MIPEP); Chew A et al.; The mitochondrial intermediate peptidase of Saccharomyces cerevisiae (YMIP) is a component of the yeast mitochondrial protein import machinery critically involved in the biogenesis of the oxidative phosphorylation (OXPHOS) system . This leader peptidase removes specific octapeptides from the amino terminus of nuclear-encoded OXPHOS subunits and components of the mitochondrial genetic apparatus . To address the biologic role of the human peptidase {MIPEP gene, HMIP polypeptide}, we have initiated its molecular and functional characterization . A full-length cDNA was isolated by screening a human liver library using a rat MIP (RMIP) cDNA as a probe . The encoded protein contained a typical mitochondrial leader peptide and showed 92 and 54% homology to RMIP and YMIP, respectively . A survey of human mitochondrial protein precursors revealed that, similar to YMIP, HMIP is primarily involved in the maturation of OXPHOS-related proteins . Northern analysis showed that the MIPEP gene is differentially expressed in human tissues, with the highest levels of expression in the heart, skeletal muscle, and pancreas, three organ systems that are frequently affected in OXPHOS disorders . Using fluorescence in situ hybridization, the MIPEP locus was assigned to 13q12 . This information offers the possibility of testing the potential involvement of HMIP in the pathophysiology of nuclear-driven OXPHOS disorders.

J Immunol, 1997 Mar 15, 158(6), 2736 - 44
HAX-1, a novel intracellular protein, localized on mitochondria, directly associates with HS1, a substrate of Src family tyrosine kinases; Suzuki Y et al.; Cross-linking of the Ag receptors on lymphocytes initiates activation of the receptor-coupled tyrosine kinases . HS1 is one of the substrates of these kinases and has been shown to transduce the signals for both clonal expansion and deletion in lymphoid cells . To gain further insight into the mechanism of action of HS1, we have tried to identify a protein that interacts with HS1 by yeast two-hybrid screening . The isolated cDNA, designated HAX-1, encodes a novel 35-kDa protein . The HAX-1 gene is expressed ubiquitously among tissues, and its protein is localized mainly in mitochondria, but also in endoplasmic reticulum and nuclear envelope in the cell . HS1/HAX-1 association is confirmed by coimmunoprecipitation of these proteins in the lysates of B lymphoma cells and COS-7 cells transfected with the corresponding cDNA expression vectors . Colocalization of these proteins in the cell is evident under confocal laser scanning microscope . Deletion mutant analysis of these proteins reveals that the association is mediated by the amino terminal region of HS1 and the carboxyl-terminal half of HAX-1 . The potential role of the HAX-1/HS1 complex is discussed.

J Mol Biol, 1997 Mar 14, 266(5), 950 - 62
A dynamic in vivo view of the HIV-I Rev-RRE interaction; Charpentier B et al.; The export of pre-mRNAs coding for the structural genes of the human immunodeficiency virus type I depends on the interaction of the Rev protein with a highly structured viral RNA sequence, the Rev-responsive element (RRE) . To gain information about the structure of the RRE and the determinants of the in vivo RRE-Rev interaction, we have analyzed the structure of the 351 nt RRE RNA within living yeast (Saccharomyces cerevisiae) by dimethyl sulfate probing with or without Rev . The in vivo structure in the absence of Rev is generally similar to the previously established solution structure . In addition, we observe a single hypermethylated guanine residue (G128), located within the Rev high-affinity binding site, in vitro as well as in vivo . The important homopurine interaction between residues 129 and 106 is required for the hyperreactivity, confirming its biological relevance . Expression of wild-type Rev leads to a protection of this region and to modifications of the RRE structure: the high-affinity site becomes further structured, and Stem IIA is destabilized . High-level expression of the oligomerization-defective mutant M4 protein leads to the same protections without destabilization of Stem IIA . Taken together with other observations, the data suggest that Rev captures the unusual conformation of the high-affinity site, followed by additional changes in the structure of the RRE.

J Biol Chem, 1997 Mar 14, 272(11), 7352 - 9
Ho endonuclease cleaves MAT DNA in vitro by an inefficient stoichiometric reaction mechanism; Jin Y et al.; Mating type switching in Saccharomyces cerevisiae initiates when Ho endonuclease makes a double-stranded DNA break at the yeast MAT locus . In this report, we characterize the fundamental biochemical properties of Ho . Using an assay that monitors cleavage of a MAT plasmid, we define an optimal in vitro reaction, showing in particular that the enzyme has a stringent requirement for zinc ions . This suggests that zinc finger motifs present in Ho are important for cleavage . The most unexpected feature of Ho, however, is its extreme inefficiency . Maximal cleavage occurs when Ho is present at a concentration of 1 molecule/3 base pairs of substrate DNA . Even under these conditions, complete digestion requires >2 h . This inefficiency results from two characteristics of Ho . First, Ho recycles slowly from cleaved product to new substrate, in part because the enzyme has an affinity for one end of its double strand break product . Second, high levels of cleavage in the in vitro reaction correlate with the appearance of large protein-DNA aggregates . At optimal Ho concentrations, these latter aggregates, referred to as "florettes," have an ordered structure consisting of a densely staining central region and loops of radiating DNA . These unusual properties may indicate that Ho plays a role in other aspects of mating type switching subsequent to double strand break formation.

J Biol Chem, 1997 Mar 14, 272(11), 7122 - 6
SUG1, a putative transcriptional mediator and subunit of the PA700 proteasome regulatory complex, is a DNA helicase; Fraser RA et al.; Yeast SUG1 was originally characterized as a transcriptional mediator for the GAL4 transactivator . A similar role in vertebrates was suggested by the ligand-enhanced interaction between mammalian homologues of yeast SUG1 and the ligand-dependent activating domain (AF-2) of nuclear receptors . SUG1 was also shown to be a component of the PA700 regulatory complex of the 26 S proteasome and a member of a large family of putative ATPases . However, no catalytic function has yet been attributed to SUG1 . We show here that SUG1 is a 3'-5' DNA helicase whose activity is dependent on an intact ATP binding domain . The sedimentation heterogeneity of mammalian SUG1 suggests that it may be associated with distinct protein complexes and therefore play multiple roles.

FEBS Lett, 1997 Mar 10, 404(2-3), 275 - 8
The plant homologue of the defender against apoptotic death gene is down-regulated during senescence of flower petals; Orzaez D et al.; Petal senescence is an example of a highly reproducible cell death programme . In this programme, DNA is fragmented internucleosomally and cells with condensed nuclei containing an increased number of 5' ends can be detected with the TUNEL technique . The pea homologue of defender against apoptotic death (dad), a gene described to suppress endogenous programmed cell death in Caenorhabditis elegans and mammals was isolated . Expression studies show that dad declines dramatically upon flower anthesis disappearing in senescent petals, and is down-regulated by the plant hormone ethylene.

FEBS Lett, 1997 Mar 10, 404(2-3), 135 - 9
Identification and characterization of homologues of the Exocyst component Sec10p; Guo W et al.; The SEC10 gene product is a member of the Exocyst complex essential for exocytosis in the budding yeast Saccharomyces cerevisiae . We report here the cloning and characterization of human Sec10p (hSec10p; GenBank accession number U85946) . hSec10p is a 77-kDa protein with 23% amino acid identity to yeast Sec10p and 37% identity to a C . elegans protein found in the database . Northern and Western blot analyses indicate that hSec10 has a broad tissue distribution . Immunofluorescence staining of COS cells cotransfected with hSec10p and a mammalian Sec8p demonstrates that these two proteins have an identical distribution in the cell including a localization in the peripheral cytoplasm . These data suggest that hSec10p is a component of the mammalian counterpart of the yeast Exocyst complex essential for post-Golgi traffic.

J Biol Chem, 1997 Mar 7, 272(10), 6510 - 8
Metaxin is a component of a preprotein import complex in the outer membrane of the mammalian mitochondrion; Armstrong LC et al.; Metaxin, a novel gene located between the glucocerebrosidase and thrombospondin 3 genes in the mouse, is essential for survival of the postimplantation mouse embryo . In this study, the subcellular location, domain structure, and biochemical function of metaxin were investigated . Anti-recombinant metaxin antibodies recognized 35- and 70-kDa proteins in mitochondria from various tissues; the 35-kDa protein is consistent in size with the predicted translation product of metaxin cDNA . When metaxin cDNA was transfected into COS cells, immunofluorescence staining demonstrated that the protein is located in mitochondria . Metaxin contains a putative mitochondrial outer membrane signal anchor domain at its C terminus, and a truncated form of metaxin lacking this signal anchor domain had a reduced association with mitochondria . In addition, metaxin was highly susceptible to proteases in intact mitochondria . We therefore conclude that metaxin is a mitochondrial protein that extends into the cytosol while anchored into the outer membrane at its C terminus . In its N-terminal region, metaxin shows significant sequence identity to Tom37, a component of the outer membrane portion of the mitochondrial preprotein translocation apparatus in Saccharomyces cerevisiae, but important structural differences, including apparently different mechanisms of targeting to membranes, also exist between the two proteins . Given the similar subcellular locations of metaxin and Tom37, the possible role of metaxin in mitochondrial preprotein import was investigated . Antibodies against metaxin, when preincubated with mitochondria, partially inhibited the uptake of radiolabeled preadrenodoxin into mitochondria . Metaxin is therefore the second mammalian component of the protein translocation apparatus of the mitochondrial outer membrane to be characterized at the molecular level and the first for which an inherited mutation has been described . The early embryonic lethal phenotype of mice lacking metaxin demonstrates that efficient import of proteins into mitochondria is crucial for cellular survival . The characterization of metaxin provides an opportunity to elucidate similarities and possible differences in the mechanisms of protein import between fungi and mammals and in the phenotypes of fungi and mammals lacking mitochondrial import receptors.

J Biol Chem, 1997 Mar 7, 272(10), 6303 - 10
Assembly of functional replication factor C expressed using recombinant baculoviruses; Podust VN et al.; Replication factor C (RF-C), a complex of five subunits, is an essential eukaryotic protein involved in both DNA replication and DNA repair . To generate an easily accessible source of human RF-C for biochemical and genetic studies, we cloned the cDNAs of all five subunits into baculoviruses so that each subunit could be expressed both as a non-fused polypeptide and as an N-terminal His-tagged fusion (-his) . Co-infection of insect cells with five baculoviruses encoding individual RF-C subunits (p140, p40, p38, p37, and p36) yielded a protein preparation active in two assays characteristic for authentic RF-C: stimulation of DNA polymerase delta DNA synthesis on singly primed single-stranded DNA template and formation of a complex of proliferating cell nuclear antigen with circular double-stranded DNA . Functional recombinant RF-C containing p40-his, p37-his, or p36-his was isolated using affinity resin . Active RF-C was reconstituted only by co-expression of all five subunits . A model for assembly of RF-C from individual subunits was derived from co-purification experiments performed with various combinations of His-tagged and non-fused subunits expressed by co-infection of insect cells with recombinant baculoviruses . p37 and p36 are proposed to form the first intermediate, which, upon addition of either p40 or p38, generates stable tertiary complexes: p40.p37.p36 and p38.p37.p36 . The remaining fourth small subunit binds to the tertiary complex to form a quaternary complex p40.p38 . p37.p36 . Large subunit p140 binds last to form the five-subunit protein.

J Biol Chem, 1997 Mar 7, 272(10), 6187 - 93
Identification of a mammalian Golgi Sec1p-like protein, mVps45; Tellam JT et al.; Our understanding of lysosomal biogenesis and general vesicular transport in animal cells has been greatly enhanced by studies of vacuolar biogenesis in yeast . Genetic screens have identified a number of proteins that play direct roles in the proper sorting of vacuolar hydrolases . These include t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and Sec1p-like proteins, which have recently been implicated as key regulators of vesicle fusion . In this study we have extended these observations in yeast and have isolated and characterized a novel member of the Sec1p-like family of proteins from mammalian cells, mVps45 . mVps45 shares a high level of identity with the Saccharomyces cerevisiae Sec1p-like protein Vps45p that is believed to function with the t-SNARE Pep12p in the fusion of Golgi-derived transport vesicles with a prevacuolar compartment . We found that mVps45 is a ubiquitously expressed peripheral membrane protein that localized to perinuclear Golgi-like and trans-Golgi network compartments in Chinese hamster ovary cells . We found that mVps45 could bind specifically to yeast Pep12p and to the mammalian Pep12p-like protein, syntaxin 6, in vitro.

Science, 1997 Mar 7, 275(5305), 1468 - 71
Requirement of the DEAD-Box protein ded1p for messenger RNA translation; Chuang RY et al.; The DED1 gene, which encodes a putative RNA helicase, has been implicated in nuclear pre-messenger RNA splicing in the yeast Saccharomyces cerevisiae . It is shown here by genetic and biochemical analysis that translation, rather than splicing, is severely impaired in two newly isolated ded1 conditional mutants . Preliminary evidence suggests that the protein Ded1p may be required for the initiation step of translation, as is the distinct DEAD-box protein, eukaryotic initiation factor 4A (eIF4A) . The DED1 gene could be functionally replaced by a mouse homolog, PL10, which suggests that the function of Ded1p in translation is evolutionarily conserved.

Oncogene, 1997 Mar 6, 14(9), 1067 - 74
The RAR alpha-PLZF chimera associated with Acute Promyelocytic Leukemia has retained a sequence-specific DNA-binding domain; Sitterlin D et al.; In most cases, Acute Promyelocytic Leukemia (APL) is associated with t(15;17) translocation which juxtaposes sequences from PML and retinoic acid receptor alpha (RAR alpha) genes . The generated PML-RAR alpha fusion interferes with wild type RAR alpha-mediated transcription and disrupts subnuclear compartments, known as PML bodies . Both defects are corrected by all trans retinoic acid (ATRA) therapy which induces differentiation of leukemic cells and clinical remission . In a rare APL syndrome associated with t(11;17), fusion of the RAR alpha gene with the PLZF gene, encoding a Zinc-finger protein produces two reciprocal RAR alpha chimeras . Although PLZF-RAR alpha and PML-RAR alpha are similar in their apparent dominant negative effects, t(11;17)-associated APL is refractory to ATRA therapy . In a yeast two-hybrid genetic screening, we isolated clones encoding the GAL4 transactivation domain fused to various parts of PLZF . Using these autonomously transactivating hybrids, similar in structure to the RAR alpha-PLZF fusion, we mapped the DNA-binding domain of PLZF to the last five Zinc-fingers, a region retained in RAR alpha-PLZF chimera and characterized a specific PLZF target sequence . Our data support the hypothesis that RAR alpha-PLZF chimera is not an inert product of reciprocal translocation and may thus contribute to ATRA unresponsiveness of t(11;17)-associated APL.

Int J Cancer, 1997 Mar 4, 70(5), 582 - 6
Expression of transporter associated with antigen processing 1 and 2 (TAP1/2) in malignant melanoma cell lines; Thor Straten P et al.; TAP1 and TAP2 molecules are involved in the transport of peptides prior to their association with class I molecules and are mandatory for efficient antigen presentation . To investigate whether loss of expression of TAP1 or TAP2 is a likely mechanism of immune escape in malignant melanoma, TAP1 and TAP2 mRNA was analyzed by RT-PCR in 39 melanoma cell lines expressing at least 2 of the known melanoma-associated antigens, tyrosinase, Melan-A/MART-1, gp100, MAGE-1 and MAGE-3 . All 39 cell lines expressed both TAP1 and TAP2 at the mRNA level . To investigate other factors potentially involved in immune escape, the expression of LMP2, LMP7, HLA class I molecules, beta2-microglobulin (beta2m) and specific HLA-A alleles was evaluated by RT-PCR and FACS analyses . All 39 cell lines expressed LMP2, LMP7 and beta2m . A single cell line (FM37) had lost the expression of class I molecules, and this same cell line showed loss of expression of the HLA-A2 heavy chain . No cell lines showed loss of expression of the HLA-A1 heavy chain . Based on our studies of in vitro established cell lines, loss of TAP1/2 or LMP2/7 expression does not appear to be a common mechanism of immune escape in malignant melanoma.

Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1692 - 7
Chirality of DNA trefoils: implications in intramolecular synapsis of distant DNA segments; Shaw SY et al.; We show that supercoiling of a DNA trefoil, the simplest knotted ring, perturbs differently the spatial writhe of its two chiral forms . As a consequence, the negative-noded and positive-noded DNA trefoils can be resolved by gel electrophoresis . Analysis of the chirality of trefoils produced by cyclization of two linear DNAs demonstrates that the two chiral trefoils are produced in equal amounts, suggesting that these DNAs do not prefer intrinsic writhe of one chirality or the other . In contrast, knotting of nicked DNA rings by a molar excess of Saccharomyces cerevisiae DNA topoisomerase II produces more negative-noded than positive-noded trefoils, indicating an asymmetry in the interaction between the enzyme and DNA crossovers of different signs . These results suggest that asymmetry in DNA crossovers and intrinsic or ligand-induced writhe in a DNA might be detectable from an analysis of trefoil chirality.

EMBO J, 1997 Mar 3, 16(5), 1093 - 102
Cloning and characterization of p52, the fifth subunit of the core of the transcription/DNA repair factor TFIIH; Marinoni JC et al.; TFIIH is a multiprotein factor involved in transcription and DNA repair and is implicated in DNA repair/transcription deficiency disorders such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy . Eight out of the nine genes encoding the subunits forming TFIIH have already been cloned . We report here the identification, cDNA cloning and gene structure of the 52 kDa polypeptide and its homology with the yeast counterpart TFB2 . This protein, along with p89/XPB, p62, p44 and p34, forms the core of TFIIH . Moreover, using in vitro reconstituted transcription and nucleotide excision repair (NER) assays and microinjection experiments, we demonstrate that p52 is directly involved in both transcription and DNA repair mechanisms in vitro and in vivo.

EMBO J, 1997 Mar 3, 16(5), 1009 - 22
CREB is activated by UVC through a p38/HOG-1-dependent protein kinase; Iordanov M et al.; Changes in environmental conditions such as the addition of growth factors or irradiation of cells in culture first affect immediate response genes . We have shown previously that short wavelength UV irradiation (UVC) elicits massive activation of several growth factor receptor-dependent pathways . At the level of the immediate response gene c-fos, these pathways activate the transcription factor complex serum response factor (SRF)-p62TCF which mediates part of the UV-induced transcriptional response . These studies have, however, suggested that more that one pathway is required for full UV responsiveness of c-fos . Using appropriate promoter mutations and dominant-negative cAMP response element (CRE)-binding protein (CREB), we now find that UVC-induced transcriptional activation depends also on the CRE at position -60 of the c-fos promoter and on the functionality of a CREB . Upon UV irradiation, CREB and ATF-1 are phosphorylated at serines 133 and 63, respectively, preceded by and dependent on activation of p38/RK/HOG-1 and of a p38/RK/HOG-1-dependent p108 CREB kinase . Although p90RSK1 and MAPKAP kinase 2 are also activated by UV, p90RSK1 does not, at least not decisively, participate in this signalling pathway to CREB and ATF-1 as it is not p38/RK/HOG-1 dependent, and CREB is a poor substrate for MAPKAP kinase 2 in vitro . On the basis of resistance to the growth factor receptor inhibitor suramin and of several types of cross-refractoriness experiments, the UVC-induced CREB/ATF-1 phosphorylation represents an as yet unrecognized route of UVC-induced signal transduction, independent of suramin-inhibitable growth factor receptors and different from the Erk 1,2-p62TCF pathway.

Cell Stress Chaperones, 1997 Mar, 2(1), 41 - 9
The protein kinase inhibitor SB203580 uncouples PMA-induced differentiation of HL-60 cells from phosphorylation of Hsp27; Schultz H et al.; HL-60 cells are an attractive model for studies of human myeloid cell differentiation . Among the well-examined parameters correlated to differentiation of HL-60 cells are the expression and phosphorylation of the small heat shock protein Hsp27 . Here we demonstrate that PMA treatment of HL-60 cells stimulates different MAP kinase cascades, leading to significant activation of ERK2 and p38 reactivating kinase (p38RK) . Using the protein kinase inhibitor SB 203580, we specifically inhibited p38RK and, thereby, activation of its target MAP kinase-activated protein kinase 2 (MAPKAP kinase 2), which is the major enzyme responsible for small Hsp phosphorylation . As a result, PMA-induced Hsp27 phosphorylation is inhibited in SB 203580-treated HL-60 cells indicating that p38RK and MAPKAP kinase 2 are components of the PMA-induced signal transduction pathway leading to Hsp27 phosphorylation . We further demonstrate that, although PMA-induced phosphorylation is inhibited, SB 203580-treated HL-60 cells are still able to differentiate to the macrophage-like phenotype as judged by decrease in cell proliferation, induction of expression of the cell surface antigen CD11b and changes in cell morphology . These results indicate that, although correlated, Hsp27 phosphorylation is not required for HL-60 cell differentiation . However, the results do not exclude that increased Hsp27 expression is involved in HL-60 cell differentiation.

Mol Biol Rep, 1997 Mar, 24(1-2), 39 - 44
The 26S-proteasome: regulation and substrate recognition; Dawson S et al.; There is extensive reprogramming of the ATPase regulators of the 26S proteasome before the programmed elimination of the abdominal intersegmental muscles (ISM) after eclosion in Manduca sexta {1} . This extensive ATPase reprogramming only occurs in ISM which are destined to die and not in flight muscle (FM) . The MS73 ATPase also increases in the proleg retractor muscles which die at a developmentally different stage to ISM . The non-ATPase regulator S5a shows a similar increase to the ATPase regulators . We have cloned the Manduca SUG2 ATPase and shown that this ATPase is a component of the 26S proteasome . This ATPase shows a similar increase in concentration to the other ATPases in 26S proteasomes before muscle death . The SUG2 ATPase is also associated with other smaller complexes besides the 26S proteasome which act as activators of the 26S proteasome . Finally, in a yeast two-hybrid genetic screen we have identified a protein in human brain which interacts with the MS73 ATPase (and human S6) . The interacting protein contains 6 ankyrin repeats and is co-immunoprecipitated with anti-MS73 antiserum after in vitro transcription/translation . The ankyrin repeat protein may interact with the MS73 ATPase as part of the substrate recognition process by the 26S proteasome . Many proteins degraded by the 26S proteasome contain ankyrin repeats, e.g . IkB and some cyclins: binding through ankyrin repeats to an ATPase regulator may complement protein ubiquitination and S5a binding as recognition signals by the 26S proteasome.

Int J Biochem Cell Biol, 1997 Mar, 29(3), 415 - 35
Growth factor-dependent phosphoinositide signalling; Hsuan JJ et al.; A wide variety of messages, in the form of diffusible growth factors, hormones and cytokines, are carried throughout multicellular organisms to coordinate important physiological properties of target cells, such as proliferation, differentiation, migration, apoptosis and metabolism . Most messengers bind to cognate receptors on target cells, which initiate a characteristic cascade of reactions within the cell, ultimately leading to the desired response . The cellular response is defined by the combination of signalling components whose individual activity depends upon the number and type of surface receptors . Consequently the responses of different cell types to one or more stimuli can be quite disparate . A molecular understanding of the signalling pathways employed by each type of receptor therefore underlies the ability to rationalize many cellular functions and to correct disfunctions . As a well studied example of the primary signalling events that take place on the cytoplasmic leaflet of the plasma membrane following receptor activation, we will discuss how the widely expressed receptor for epidermal growth factor (EGF) causes the phosphorylation and hydrolysis of a signalling precursor, the membrane lipid phosphatidylinositol . This paradigm will be used to illustrate certain general principles of signalling, including formation of multienzyme complexes, compartmentation of second messengers and intermediates, and cross-talk between different signalling pathways.

Electrophoresis, 1997 Mar-Apr, 18(3-4), 324 - 7
Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients; Sanchez JC et al.; A simple and inexpensive methacrylate rehydration chamber was built to accommodate ten immobilized pH gradient (IPG) strips . In the chamber, entire IPG gels were used for sample application, with the protein entering the gels during their rehydration . For rehydration, commercially available or laboratory-made strips were positioned in the grooves with the gel in contact with 500 microL of sample for 6 h or overnight . This avoided the use of sample cups, eliminated precipitation at the sample application site, thus improving resolution over the entire pH range of the gels . It also allowed precise control of protein amounts and sample volumes loaded into the IPG gels, and also lowered costs of reagents during rehydration and equilibration owing to the reduced volumes . Up to 5 mg of protein can be loaded on wide IPG gels and up to 15 mg of some samples on narrow pH range gels.

J Exp Clin Cancer Res, 1997 Mar, 16(1), 3 - 10
BCL-2: the pendulum of the cell fate; Blandino G et al.; The homeostasis of normal tissues is a balance between cell proliferation and cell death . Alterations of both pathways contribute to a clonal expansion of cancer cells . Bcl-2 and its family play an important role in the regulation of the apoptotic pathway . Apoptosis or programmed cell death is an active form of cell suicide that is characterized by specific morphological and biochemical events . These include cleavege of genomic DNA into oligonucleosome-length DNA fragments by endonucleases, chromatin condensation and marginalization, nuclear fragmentation, plasma membrane blebbing, and cell shrinkage . Though the role of apoptosis is clearly defined in the maintaining of physiological tissue homeostasis, several pathological conditions and external factors cause apoptosis . Anticancer drugs and radiation, two of the most important tools in the cancer treatment, cause apoptotic cell death . The understanding of the mechanisms underlying the regulation of the apoptosis in response to different types of DNA damage might provide relevant information to improve cancer treatment . In this review we mainly discuss bcl-2 and its partners in human cancers and how their disregulation might contribute to the development and the difficult treatment of cancer.

Hum Mol Genet, 1997 Mar, 6(3), 409 - 16
Immunocytochemical localization of the Menkes copper transport protein (ATP7A) to the trans-Golgi network; Dierick HA et al.; We have generated polyclonal antibodies against the amino-terminal third of the Menkes protein (ATP7A; MNK) by immunizing rabbits with a histidine-tagged MNK fusion construct containing metal-binding domains 1-4 . The purified antibodies were used in Western analysis of cell lysates and in indirect immunofluorescence experiments on cultured cells . On Western blots, the antibodies recognized the approximately 165 kDa MNK protein in CHO cells and human fibroblasts . No MNK signal could be detected in fibroblasts from a patient with Menkes disease or in Hep3B hepatocellular carcinoma cells, confirming the specificity of the antibodies . Immunocytochemical analysis of CHO cells and human fibroblasts showed a distinct perinuclear signal corresponding to the pattern of the Golgi complex . This staining pattern was similar to that of alpha-mannosidase II which is a known resident enzyme of the Golgi complex . Using brefeldin A, a fungal inhibitor of protein secretion, we further demonstrated that the MNK protein is localized to the trans-Golgi network . This data provides direct evidence for a subcellular localization of the MNK protein which is similar to the proposed vacuolar localization of Ccc2p, the yeast homolog of MNK and WND (ATP7B), the Wilson disease gene product . In light of the proposed role of MNK both in subcellular copper trafficking and in copper efflux, these data suggest a model for how these two processes are linked and represent an important step in the functional analysis of the MNK protein.

Biophys J, 1997 Mar, 72(3), 1022 - 30
Probing protein hydration and conformational states in solution; Reid C et al.; The addition of polyethylene glycol (PEG), of various molecular weights, to solutions bathing yeast hexokinase increases the affinity of the enzyme for its substrate glucose . The results can be interpreted on the basis that PEG acts directly on the protein or indirectly through water activity . The nature of the effects suggests to us that PEG's action is indirect . Interpretation of the results as an osmotic effect yields a decrease in the number of water molecules, delta Nw, associated with the glucose binding reaction . delta Nw is the difference in the number of PEG-inaccessible water molecules between the glucose-bound and glucose-free conformations of hexokinase . At low PEG concentrations, delta Nw increases from 50 to 326 with increasing MW of the PEG from 300 to 1000, and then remains constant for MW-PEG up to 10,000 . This suggests that up to MW 1000, solutes of increasing size are excluded from ever larger aqueous compartments around the protein . Three hundred and twenty-six waters is larger than is estimated from modeling solvent volumes around the crystal structures of the two hexokinase conformations . For PEGs of MW > 1000, delta Nw falls from 326 to about 25 waters with increasing PEG concentration, i.e., PEG alone appears to "dehydrate" the unbound conformation of hexokinase in solution . Remarkably, the osmotic work of this dehydration would be on the order of only one k T per hexokinase molecule . We conclude that under thermal fluctuations, hexokinase in solution has a conformational flexibility that explores a wide range of hydration states not seen in the crystal structure.

Leuk Lymphoma, 1997 Mar, 25(1-2), 23 - 35
Inactivation of cyclin-dependent kinase inhibitor genes and development of human acute leukemias; Ragione FD et al.; A large body of evidence has definitely demonstrated that cancer development and/or progression is strictly linked to alterations of molecular mechanisms controlling the cell division cycle . In particular, those aberrations which cause a shortening of G1 phase length and a deregulated S phase entry seem to be very important . Two main tumor suppressor loci, involved in the cell cycle regulation, are frequently altered in human tumors . One is located on 13q14 chromosome and includes the gene coding pRb protein while the other is located on 9p21 chromosome and involves two genes, namely p16INK4A and p15INK4B which belong to the same gene family . While RB1 gene is scarcely altered in hematological tumors, the putative tumor suppressor gene(s) on 9p21 appear(s) to be frequently inactivated in some subtypes of cancers derived from hematopoietic tissues . This manuscript will review the main biochemical aspects of the cell division cycle with major emphasis devoted to the findings regarding the recently characterized small proteic mitotic inhibitors and to their possible role in cancer formation . Particular attention will be paid to the data concerning the incidence of p16INK4A (and p15INK4B) gene(s) inactivation in human acute lymphoblastic leukemias . Indeed, such gene(s) seems to be the main, and until now the unique, tumor suppressor gene consistently altered in this acute hematological cancer diseases . Finally, future directions in studies on the connection between cell cycle control and leukemogenesis will be analyzed.

Am J Physiol, 1997 Mar, 272(3 Pt 1), C923 - 30
Na+-K+-ATPase alpha-subunit containing Q905-V930 of gastric H+-K+-ATPase alpha preferentially assembles with H+-K+-ATPase beta; Wang SG et al.; Amino acids N886-A911 of the rat Na+-K+-ATPase alpha3-subunit were replaced by the corresponding region (Q905-V930) of the rat gastric H+-K+-ATPase alpha-subunit . The chimera (NGH26) was expressed in yeast with the rat Na+-K+ -ATPase beta1-subunit (rbeta1), the rat H+-K+-ATPase beta-subunit (HKbeta), the chimeric beta-subunit NHbeta1 (containing the carboxy-terminal ectodomain of HKbeta), or the chimeric beta-subunit HNbeta1 (containing the carboxy-terminal ectodomain of rbeta1) . Increased resistance to trypsin digestion indicated that NGH26 preferentially assembled with HKbeta and NHbeta1 rather than with rbeta1 or HNbeta1 . Ouabain binding also indicated that more functional complexes were assembled when NGH26 was expressed with HKbeta or NHbeta1 . These results suggest that the sequence Q905-V930 interacts with the HKbeta-subunit on the extracellular side of the cell membrane . The NGH26 + HKbeta complex is less stable than alpha3 + HKbeta when heated and also has a lower binding affinity for ouabain {dissociation constant (Kd) = 63 nM} compared with alpha3 + rbeta1 or alpha3 + HKbeta (K(d) = 5-10 nM) . In contrast, the NGH26+NHbeta1 complex is thermally as stable as alpha3 + rbeta1 complexes, and its ouabain binding affinity (K(d) = 10 nM) is the same as the wild type . These results indicate that the amino acids Q905-V930 of the rat gastric H+-K+-ATPase alpha-subunit preferentially associate with the extracellular domain of H+-K+-ATPase beta-subunit to form functional pump complexes and that the cytoplasmic and/or transmembrane region of the beta-subunit influences the stability of the alpha beta complexes.

Bull Math Biol, 1997 Mar, 59(2), 339 - 97
Generic properties of combinatory maps: neutral networks of RNA secondary structures; Reidys C et al.; Random graph theory is used to model and analyse the relationships between sequences and secondary structures of RNA molecules, which are understood as mappings from sequence space into shape space . These maps are non-invertible since there are always many orders of magnitude more sequences than structures . Sequences folding into identical structures form neutral networks . A neutral network is embedded in the set of sequences that are compatible with the given structure . Networks are modeled as graphs and constructed by random choice of vertices from the space of compatible sequences . The theory characterizes neutral networks by the mean fraction of neutral neighbors (lambda) . The networks are connected and percolate sequence space if the fraction of neutral nearest neighbors exceeds a threshold value (lambda > lambda *) . Below threshold (lambda < lambda *), the networks are partitioned into a largest "giant" component and several smaller components . Structures are classified as "common" or "rare" according to the sizes of their pre-images, i.e . according to the fractions of sequences folding into them . The neutral networks of any pair of two different common structures almost touch each other, and, as expressed by the conjecture of shape space covering sequences folding into almost all common structures, can be found in a small ball of an arbitrary location in sequence space . The results from random graph theory are compared to data obtained by folding large samples of RNA sequences . Differences are explained in terms of specific features of RNA molecular structures.

Neuron, 1997 Mar, 18(3), 453 - 61
DOC2 proteins in rat brain: complementary distribution and proposed function as vesicular adapter proteins in early stages of secretion; Verhage M et al.; DOC2 proteins constitute a novel protein family that may function in secretion and contain a double C2 domain . We have cloned and characterized two DOC2 isoforms in rat brain and studied their interactions with other proteins implicated in secretion . DOC2A was virtually brain specific, DOC2B ubiquitous . Within brain, the isoforms were expressed nonuniformly and complementary within neurons, not astroglia, and copurified with synaptic vesicles . Affinity purification, yeast two-hybrid analysis, and coimmunoprecipitation revealed that DOC2 binds munc18, a protein also implicated in secretion . The first DOC2 C2 domain and most of munc18 are involved in direct interactions . Munc18 may regulate formation of 'core complexes' during vesicle docking, by interacting with syntaxin . We show that DOC2 and syntaxin compete for munc18 . Other core complex components shifted the equilibrium between syntaxin-munc18 versus DOC2-munc18 . These data suggest that DOC2 proteins are vesicular adapter proteins regulating munc18-syntaxin complexes and herewith synaptic vesicle docking.

Mol Carcinog, 1997 Mar, 18(3), 171 - 6
Reappraisal of p53 mutations in human malignant astrocytic neoplasms by p53 functional assay: comparison with conventional structural analyses; Tada M et al.; We previously reported clonal expansion of p53 mutations in malignant astrocytic tumors detected with a yeast p53 functional assay that measures mutant p53 alleles quantitatively and loss of p53 transcriptional competence qualitatively (Tada et al., Int J Cancer 67:447-450, 1996) . This method selectively detects inactivating mutations and is relatively insensitive to contamination of tumor samples with normal tissue . To determine whether the mutation frequency and spectrum detected in this way differ from those seen with conventional techniques, 54 malignant astrocytomas were tested with the yeast assay, and the abnormalities detected were characterized by DNA sequencing . Inactivating p53 mutations were found in 67% of anaplastic astrocytomas and 41% of glioblastomas . Overall, mutations were found in 48% of tumors, compared with only 29% in previous studies (P < 0.005), a difference that probably reflects the greater sensitivity of the yeast assay than of conventional techniques . The frequency of mutations in anaplastic astrocytomas (in our study plus published studies) was significantly higher than in glioblastomas (39% vs 29%; P < 0.05) . This suggests that acquisition of p53 mutations is not rate limiting for progression to glioblastoma and that many glioblastomas develop by p53-independent pathways . Sequencing of mutant p53 cDNAs rescued from yeast showed that the mutation spectrum for functionally inactive mutants was nearly identical to the spectra from previous studies on structural mutants, indicating that transcriptional activity is the critical biological target of p53 mutation in malignant astrocytomas.

Plant J, 1997 Mar, 11(3), 605 - 12
A glucocorticoid-mediated transcriptional induction system in transgenic plants; Aoyama T et al.; A novel chemical induction system for transcription in plants has been developed, taking advantage of the regulatory mechanism of vertebrate steroid hormone receptors . A chimeric transcription of the DNA-binding domain of the yeast transcription factor GAL4, the transactivating domain of the herpes viral protein VP16, and the receptor domain of the rat glucocorticoid receptor (GR) . The GVG gene was introduced into transgenic tobacco and Arabidopsis together with a luciferase (Luc) gene which was transcribed from a promoter containing six tandem copies of the GAL4 upstream activating sequence . Induction of luciferase activity was observed when the transgenic tobacco plants were grown on an agar medium containing dexamethasone (DEX), a strong synthetic glucocorticoid . Induction levels of the luciferase activity were well correlated with DEX concentrations in the range from 0.1 to 10 microM and the maximum expression level was over 100 times that of the basal level . Analysis of the induction kinetics by Northern blot analysis showed that the Luc mRNA was first detected 1 h after DEX treatment and increased to the maximum level in 4 h . The stationary induction level and the duration of the induction varied with the glucocorticoid derivative used . The GVG gene activity can also be regulated by DEX in transgenic Arabidopsis plants . The results indicate that a stringent chemical control of transcription can be achieved in plants with the GVG system . Advantages and potential uses of this system are also discussed.

Microbiol Mol Biol Rev, 1997 Mar, 61(1), 17 - 32
Genetic regulation of nitrogen metabolism in the fungi; Marzluf GA; In the fungi, nitrogen metabolism is controlled by a complex genetic regulatory circuit which ensures the preferential use of primary nitrogen sources and also confers the ability to use many different secondary nitrogen sources when appropriate . Most structural genes encoding nitrogen catabolic enzymes are subject to nitrogen catabolite repression, mediated by positive-acting transcription factors of the GATA family of proteins . However, certain GATA family members, such as the yeast DAL80 factor, act negatively to repress gene expression . Selective expression of the genes which encode enzymes for the metabolism of secondary nitrogen sources is often achieved by induction, mediated by pathway-specific factors, many of which have a GAL4-like C6/Zn2 DNA binding domain . Regulation within the nitrogen circuit also involves specific protein-protein interactions, as exemplified by the specific binding of the negative-acting NMR protein with the positive-acting NIT2 protein of Neurospora crassa . Nitrogen metabolic regulation appears to play a significant role in the pathogenicity of certain animal and plant fungal pathogens.

Biotechnol Prog, 1997 Mar-Apr, 13(2), 123 - 31
Estimation of the rate of cloned gene integration via retrotransposition; Wang X et al.; A theoretical method has been developed to estimate retrotransposition (integration) rates of the Saccharomyces cerevisiae Ty3 and Ty1 retrotransposons bearing heterologous and homologous genes (neor, HIS3) . The method is based on population growth modeling and Lea and Coulson's maximal likelihood method for mutation rate estimation (Lea and Coulson, 1949) . This method has allowed us to examine directly retrotransposition rates of GAL-regulated marked Ty3 and Ty1 elements into the whole yeast genome, not just a particular DNA sequence, for the purpose of cloned gene integration . The integration rates of a Ty3-neor system (ca . 1 x 10(-3) cell-1 generation-1) and a Ty1-neor system (ca . (2-3) x 10(-3) cell-1 generation-1) were not significantly affected by temperature (18 and 30 degrees C) . However, the retrotransposition rate of the Ty3-neor-HIS3 system increased from ca . 2 x 10(-5) to 2 x 10(-4) cell-1 generation-1 when the temperature was decreased from 30 to 18 degrees C . The retrotransposition rate of Ty3-neor was significantly higher than that of Ty3-neor-HIS3 and slightly lower than that of Ty1-neor . This method can be used to estimate integration rates of other Ty3 and Ty1 elements and to evaluate the efficiency of Ty-mediated cloned gene integration.

J Cell Sci, 1997 Mar, 110 ( Pt 6), 753 - 63
Cdc6p establishes and maintains a state of replication competence during G1 phase; Detweiler CS et al.; CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae . Here we examine the timing of Cdc6p expression and function during the cell cycle . Cdc6p is expressed primarily between mitosis and Start . This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven by a constitutively induced promoter . Transcriptional repression of CDC6 or exposure of cdc6-1(ts) cells to the restrictive temperature at mitosis blocks subsequent S phase, demonstrating that the activity of newly synthesized Cdc6p is required each cell cycle for DNA replication . In contrast, similar perturbations imposed on cells arrested in G(1) before Start have moderate or no effects on DNA replication . This suggests that, between mitosis and Start, Cdc6p functions in an early step of initiation, effectively making cells competent for replication . Prolonged exposure of cdc6-1(ts) cells to the restrictive temperature at the pre-Start arrest eventually does cripple S phase, indicating that Cdc6p also functions to maintain this initiation competence during G(1) . The requirement for Cdc6p to establish and maintain initiation competence tightly correlates with the requirement for Cdc6p to establish and maintain the pre-replicative complex at a replication origin, strongly suggesting that the pre-replicative complex is an important intermediate for the initiation of DNA replication . Confining assembly of the complex to G(1) by restricting expression of Cdc6p to this period may be one way of ensuring precisely one round of replication per cell cycle.

Proteins, 1997 Mar, 27(3), 438 - 49
Hydroxynitrile lyase from Hevea brasiliensis: molecular characterization and mechanism of enzyme catalysis; Hasslacher M et al.; (S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis is a 29 kDa single chain protein that catalyses the breakdown or formation of a C--C bond by reversible addition of hydrocyanic acid to aldehydes or ketones . The primary sequence of Hnl has no significant homology to known proteins . Detailed homology investigations employing PROFILESEARCH and secondary structure prediction algorithms suggest that Hnl is a member of the alpha/beta hydrolase fold protein family and contains a catalytic triad as functional residues for catalysis . The significance of predicted catalytic residues was tested and confirmed by site-directed mutagenesis and expression of mutant and wild-type proteins in the yeast, Saccharomyces cerevisiae . Based on these data we suggest a mechanistic model for the (S)-cyanohydrin synthesis catalyzed by hydroxynitrile lyase from Hevea brasiliensis.

Alcohol, 1997 Mar-Apr, 14(2), 181 - 9
Mechanism for the inhibition of aldehyde dehydrogenase by nitric oxide; DeMaster EG et al.; The inhibition of Saccharomyces cerevisiae aldehyde dehydrogenase (AlDH) by gaseous nitric oxide (NO) in solution and by NO generated from diethylamine nonoate was time and concentration dependent . The presence of oxygen significantly reduced the extent of inhibition by NO, indicating that NO itself rather than an oxidation product of NO such as N2O3 is the inhibitory species under physiological conditions . A cysteine residue at the active site of the enzyme was implicated in this inhibition based on the following observations: a) NAD+ and NADP+, but not reduced cofactors, significantly enhanced inhibition of AlDH by NO; b) the aldehyde substrate, benzaldehyde, blocked inhibition; and c) inhibition was accompanied by loss of free sulfhydryl groups on the enzyme . Activity of the NO-inactivated enzyme was readily restored by treatment with dithiothreitol (DTT), but not with GSH . This difference was attributed, in part, to a redox process leading to the formation of a cyclic DTT disulfide . Based on the chemistry deduced from model systems, the reaction of NO with AlDH sulfhydryls was shown to produce intramolecular disulfides and N2O . These disulfides were shown to be intrasubunit disulfides by nonreducing SDS-PAGE analysis of the NO- inhibited enzyme . Following complete inhibition of AlDH by NO, four of the eight titratable (Ellman's reagent) sulfhydryl groups of AlDH were found to be oxidized to disulfides . These results suggest that a) the sulfhydryl group of active site Cys-302 and a proximal cysteine are oxidized to form an intrasubunit disulfide by NO; b) only two of the four subunits of AlDH are catalytically active; and c) NO preferentially oxidizes sulfhydryl groups of the catalytically active subunits . A detailed mechanism for the inhibition of AlDH by NO is presented.

Protein Sci, 1997 Mar, 6(3), 657 - 65
Thermal denaturation of iso-1-cytochrome c variants: comparison with solvent denaturation; Herrmann LM et al.; Thermal denaturation studies as a function of pH were carried out on wild-type iso-1-cytochrome c and three variants of this protein at the solvent-exposed position 73 of the sequence . By examining the enthalpy and Tm at various pH values, the heat capacity increment (delta Cp), which is dominated by the degree of change in nonpolar hydration upon protein unfolding, was found for the wild type where lysine 73 is normally present and for three variants . For the Trp 73 variant, the delta Cp value (1.15 +/- 0.17 kcal/mol K) decreased slightly relative to wild-type iso-1-cytochrome c (1.40 +/- 0.06 kcal/mol K), while for the Ile 73 (1.65 +/- 0.07 kcal/mol K) and the Val 73 (1.50 +/- 0.06 kcal/mol K) variants, delta Cp increased slightly . In previous studies, the Trp 73, Ile 73, and Val 73 variants have been shown to have decreased m-values in guanidine hydrochloride denaturations relative to the wild-type protein (Hermann L, Bowler BE, Dong A, Caughey WS . 1995 . The effects of hydrophilic to hydrophobic surface mutations on the denatured state of iso-1-cytochrome c: Investigation of aliphatic residues . Biochemistry 34:3040-3047) . Both the m-value and delta Cp are related to the change in solvent exposure upon unfolding and other investigators have shown a correlation exists between these two parameters . However, for this subset of variants of iso-1-cytochrome c, a lack of correlation exists which implies that there may be basic differences between the guanidine hydrochloride and thermal denaturations of this protein . Spectroscopic data are consistent with different denatured states for thermal and guanidine hydrochloride unfolding . The different response of m-values and delta Cp for these variants will be discussed in this context.

Mamm Genome, 1997 Mar, 8(3), 186 - 92
Construction of a swine YAC library allowing an efficient recovery of unique and centromeric repeated sequences; Rogel-Gaillard C et al.; A swine DNA genomic library was constructed in yeast artificial chromosome (YAC) using the pYAC4 vector and the AB1380 strain . The DNA prepared from two Large White males was partially digested with EcoRI and size selected after both digestion and ligation . The YAC library contained 33792 arrayed clones with an average size of 280 kb as estimated by analysis of 2% of the clones, thus representing a threefold coverage of the swine haploid genome . The library was organized in pools to facilitate the PCR screening . The complexity of the library was tested both for unique and centromeric repeated sequences . In all, 20 out of 22 primer sets allowed the characterization of one to six clones containing specific unique sequences . These sequences are known to be on Chromosomes (Chrs) 1, 2, 5, 6, 7, 8, 13, 14, 15, 17, and X . Eight additional clones carrying centromeric repeat units were also isolated with a single primer set . The sequencing of 37 distinct repeat units of about 340 bp subcloned from these eight YACs revealed high sequence diversity indicating the existence of numerous centromeric repeat unit subfamilies in swine . Furthermore, the analysis of the restriction patterns with selected enzymes suggested a higher order organization of the repeat units . According to preliminary FISH experiments on a small number of randomly chosen YACs and YACs carrying specific sequences, the chimerism appeared to be low . In addition, primed in situ labeling experiments favored the idea that the YACs with centromeric repeat sequences were derived from a subset of metacentric and submetacentric chromosomes.

Biochem J, 1997 Mar 1, 322 ( Pt 2), 649 - 54
Comparison of long-chain fatty acyl-CoA synthetases from rabbit heart and liver: substrate preferences and effects of Mg2+; Weis MT et al.; Rabbit heart has a single, non-specific, fatty acyl-CoA synthetase (HP1) which is dependent on Mg2+, apart from the requirement for MgATP2- . Two long-chain fatty acyl-CoA synthetase activities (LP1 and LP2) can be resolved by hydroxyapatite chromatography of liver preparations; the Mg2+ requirement for these enzymes is undefined . These experiments were done to define the Mg2+ requirements of the liver enzymes and to compare them with the heart enzyme . For all three sources of enzyme and for arachidonic, oleic and palmitic acid substrates, the overall velocity of the reaction increased as {Mg2+} increased . Depending on the substrate and the source of enzyme, the increase in overall velocity could be attributed to changes in affinity or maximal velocity or both . The substrate preference of the HP1 enzyme for arachidonic acid (AA) was fifth or sixth of eight substrates regardless of the concentration of Mg2+ . In contrast, increasing {Mg2+} shifted the relative substrate preference of both liver enzymes for AA . At low {Mg2+}, AA was ranked seventh or eighth (least preferred) of eight substrates, whereas at high {Mg2+}, AA was ranked as fifth or sixth . Hill plots of competition studies were consistent with Mg2+-induced positive co-operativity in LP1, but not in HP1 or LP2 . Although enzymes from the three sources exhibit substantial kinetic differences, it is uncertain whether they are three different enzymes.

J Clin Invest, 1997 Mar 1, 99(5), 975 - 86
Common and distinct intracellular signaling pathways in human neutrophils utilized by platelet activating factor and FMLP; Nick JA et al.; Stimulation of human neutrophils with chemoattractants FMLP or platelet activating factor (PAF) results in different but overlapping functional responses . We questioned whether these differences might reflect patterns of intracellular signal transduction . Stimulation with either PAF or FMLP resulted in equivalent phosphorylation and activation of the mitogen-activated protein kinase (MAPk) homologue 38-kD murine MAP kinase homologous to HOG-1 (p38) MAPk . Neither FMLP nor PAF activated c-jun NH2-terminal MAPk (JNKs) . Under identical conditions, FMLP but not PAF, resulted in significant p42/44 (ERK) MAPk activation . Both FMLP and PAF activated MAP kinase kinase-3 (MKK3), a known activator of p38 MAPk . Both MAP ERK kinase kinase-1 (MEKK1) and Raf are activated strongly by FMLP, but minimally by PAF . Pertussis toxin blocked FMLP-induced activation of the p42/44 (ERK) MAPk cascade, but not that of p38 MAPk . A specific p38 MAPk inhibitor (SK&F 86002) blocked superoxide anion production in response to FMLP and reduced adhesion and chemotaxis in response to PAF or FMLP . These results demonstrate distinct patterns of intracellular signaling for two chemoattractants and suggest that selective activation of intracellular signaling cascades may underlie different patterns of functional responses.

Development, 1997 Mar, 124(5), 1089 - 98
Specific residues in the Pbx homeodomain differentially modulate the DNA-binding activity of Hox and Engrailed proteins; Peltenburg LT et al.; Two classes of homeodomain proteins, Hox and Engrailed, have been shown to act in concert with the atypical homeodomain proteins Pbx and extradenticle . We now show that specific residues located within the Pbx homeodomain are essential for cooperative DNA binding with Hox and Engrailed gene products . Within the N-terminal region of the Pbx homeodomain, we have identified a residue that is required for cooperative DNA binding with three Hox gene products but not for cooperativity with Engrailed-2 (En-2) . Furthermore, there are similarities between heterodimeric interactions involving the yeast mating type proteins MATa1 and MATalpha2 and those that allow the formation of Pbx/Hox and Pbx/En-2 heterodimers . Specifically, residues located in the a1 homeodomain that were previously shown to form a hydrophobic pocket allowing the alpha2 C-terminal tail to bind, are also required for Pbx/Hox and Pbx/En-2 cooperativity . Furthermore, we show that three residues located in the turn between helix 1 and helix 2, characteristic of many atypical homeodomain proteins, are required for cooperative DNA binding involving both Hox and En-2 . Replacement of the three residues located in the turn between helix 1 and helix 2 of the Pbx homeodomain with those of the atypical homeodomain proteins controlling cell fate in the basidiomycete Ustilago maydis, bE5 and bE6, allows cooperative DNA binding with three Hox members but abolishes interactions with En-2 . The data suggest that the molecular mechanism of homeodomain protein interactions that control cell fate in Saccharomyces cerevisiae and in the basidiomycetes may well be conserved in part in multicellular organisms.

Cell Growth Differ, 1997 Mar, 8(3), 283 - 91
Evidence of a retinoid signaling alteration involving the activator protein 1 complex in tumorigenic human bronchial epithelial cells and non-small cell lung cancer cells; Lee HY et al.; Retinoids, including retinol and retinoic acid derivatives, inhibit the growth of normal human bronchial epithelial (HBE) cells . Using a lung carcinogenesis model consisting of normal, immortalized, and tumorigenic HBE cells, we showed previously that, compared to normal HBE cells, the tumorigenic HBE cell line 11701 is resistant to the growth-inhibitory effects of all-trans-retinoic acid (t-RA) . Retinoid receptor function is preserved in tumorigenic 11701 cells, suggesting that other retinoid signaling components are altered . The activator protein 1 (AP-1) complex is a component of the retinoid signaling pathway and has demonstrated importance in cellular growth and differentiation . Therefore, we investigated whether AP-1 is involved in a retinoid signaling defect in tumorigenic 11701 cells and in retinoid-resistant non-small cell lung cancer (NSCLC) cell lines . We found that t-RA treatment inhibited AP-1 transcriptional activity in normal HBE cells but not in tumorigenic 11701 cells nor in the NSCLC cell lines Calu-1, Calu-6, SKMES-1, and ChaGo K1 . We sought mechanisms for this retinoid signaling alteration involving AP-1 in tumorigenic 11701 cells . Basal AP-1 transcriptional activity; AP-1 DNA-binding activity; and the mRNA levels of c-fos, the AP-1 coactivator Jun activation domain-binding protein 1, and the retinoid receptor corepressor, the silencing mediator for retinoid and thyroid hormone receptors (SMRT), were lower in tumorigenic 11701 cells than in normal HBE cells . Transient transfection of tumorigenic 11701 cells with c-fos or CREB binding protein, which is a coactivator of AP-1 and retinoid receptors, enhanced basal AP-1 transcriptional activity but did not alter the effects of t-RA on AP-1 transcriptional activity . These findings provide evidence of a retinoid signaling alteration involving AP-1 in tumorigenic 11701 and NSCLC cells . Furthermore, the inhibitory effect of t-RA on AP-1 transcriptional activity was not restored in tumorigenic 11701 cells by transfection of c-fos, silencing mediator for retinoid and thyroid hormone receptors, Jun activation domain-binding protein 1, or CREB-binding protein, suggesting the involvement of other transcriptional coregulators in this retinoid signaling defect.

Arch Biochem Biophys, 1997 Mar 1, 339(1), 2 - 8
The heterologous interactions among plant 14-3-3 proteins and identification of regions that are important for dimerization; Wu K et al.; The 14-3-3 proteins constitute a family of dimeric proteins that are involved in many cellular functions . At least two mammalian 14-3-3 proteins can form heterodimers and the approximate regions important for dimerization have been identified . In this study, we demonstrate that eight Arabidopsis and one maize 14-3-3 protein can dimerize with each other and with themselves . Native gel Western analysis of Arabidopsis cell extract also suggests the presence of 14-3-3 heterodimers in vivo . Finally, we identified the domains of one 14-3-3 protein that are sufficient for homodimerization and heterodimerization . These data support the hypothesis that evolutionarily divergent 14-3-3 proteins can interact with each other to form diverse molecular modulators or adapters in signaling pathways.

Genetics, 1997 Mar, 145(3), 605 - 14
Hyperactivation of the silencing proteins, Sir2p and Sir3p, causes chromosome loss; Holmes SG et al.; The SIRgene products maintain transcriptional repression at the silent mating type loci and telomeres in Saccharomyces cerevisiae, although no enzymatic or structural activity has been assigned to any of the Sir proteins nor has the role of any of these proteins in transcriptional silencing been clearly defined . We have investigated the functions and interactions of the Sir2, Sir3, and Sir4 proteins by overexpressing them in yeast cells . We find that Sir2p and Sir3p are toxic when overexpressed, while high Sir4p levels have no toxic effect . Epistasis experiments indicate that Sir2p-induced toxicity is diminished in strains lacking the SIR3 gene, while both Sir2p and Sir4p are required for Sir3p to manifest its full toxic effect . In addition, the effects of Sir2 or Sir3 overexpression are exacerbated by specific mutations in the N-terminus of the histone H4 gene . These results are consistent with a model in which Sir2p, Sir3p and Sir4p function as a complex and interact with histones to modify chromatin structure . We find no evidence that toxicity from high levels of the Sir proteins results from widespread repression of transcription . Instead, we find that high levels of Sir2p and/or Sir3p cause a profound decrease in chromosome stability . These results can be appreciated in the context of the effects of Sir2p in histone acetylation and of chromatin structure on chromosome stability.

Cancer Res, 1997 Mar 1, 57(5), 926 - 9
Lack of imprinting of three human cyclin-dependent kinase inhibitor genes; Cost GJ et al.; Genomic imprinting is an epigenetic modification in the germline leading to parental allele-specific gene expression in somatic cells . We have previously found that imprinted genes can be abnormally expressed or silenced in tumors and that the cyclin-dependent kinase inhibitor (CKI) CDKN1C (p57KIP2) is normally imprinted, with preferential expression of the maternal allele . Here we analyze the imprinting status of three additional CKIs, the abnormal expression and/or chromosomal localization of which has been implicated in human malignancy: CDKN1A, CDKN1B, and CDKN2C . Allele-specific expression was examined by reverse transcription-PCR, using primers that span transcribed polymorphisms as well as exon/intron boundaries, to distinguish cDNA products from genomic DNA . Biallelic expression was observed for all three genes in both fetal and adult tissues . Thus, genomic imprinting is not a generalized feature of CKIs.

Mol Cell Biol, 1997 Mar, 17(3), 1595 - 606
Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt; Kulik G et al.; We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light . Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I . Overexpression of a kinase-defective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis . The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists . However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60v-src also was protective . There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70S6 kinase . On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase) . To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis . Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling . We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinase-dead Akt was not . We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.

Mol Cell Biol, 1997 Mar, 17(3), 1484 - 9
Incubation at the nonpermissive temperature induces deficiencies in UV resistance and mutagenesis in mouse mutant cells expressing a temperature-sensitive ubiquitin-activating enzyme (E1); Ikehata H et al.; In temperature-sensitive (ts) mutants of mouse FM3A cells, the levels of mutagenesis and survival of cells treated with DNA-damaging agents have been difficult to assess because they are killed after their mutant phenotypes are expressed at the nonpermissive temperature . To avoid this difficulty, we incubated the ts mutant cells at the restrictive temperature, 39 degrees C, for only a limited period after inducing DNA damage . We used ts mutants defective in genes for ubiquitin-activating enzyme (E1), DNA polymerase alpha, and p34(cdc2) kinase . Whereas the latter two showed no effect, E1 mutants were sensitized remarkably to UV light if incubated at 39 degrees C for limited periods after UV exposure . Eighty-five percent of the sensitization occurred within the first 12 h of incubation at 39 degrees C, and more than 36 h at 39 degrees C did not produce any further sensitization . Moreover, while the 39 degrees C incubation gave E1 mutants a moderate spontaneous mutator phenotype, the same treatment significantly diminished the level of UV-induced 6-thioguanine resistance mutagenesis and extended the time necessary for expression of the mutation phenotype . These characteristics of E1 mutants are reminiscent of the defective DNA repair phenotypes of Saccharomyces cerevisiae rad6 mutants, which have defects in a ubiquitin-conjugating enzyme (E2), to which E1 is known to transfer ubiquitin . These results demonstrate the involvement of E1 in eukaryotic DNA repair and mutagenesis and provide the first direct evidence that the ubiquitin-conjugation system contributes to DNA repair in mammalian cells.

Mol Cell Biol, 1997 Mar, 17(3), 1450 - 8
Cyclic AMP-dependent protein kinase inhibits ADH2 expression in part by decreasing expression of the transcription factor gene ADR1; Dombek KM et al.; In Saccharomyces cerevisiae, the unregulated cyclic AMP-dependent protein kinase (cAPK) activity of bcy1 mutant cells inhibits expression of the glucose-repressible ADH2 gene . The transcription factor Adr1p is thought to be the primary target of cAPK . Here we demonstrate that the decreased abundance of Adr1p in bcy1 mutant cells contributes to the inhibition of ADH2 expression . Activation of ADH2 transcription was blocked in bcy1 mutant cells, and UAS1, the Adr1p binding site in the ADH2 promoter, was sufficient to mediate this effect . Concurrent with this loss of transcriptional activation was an up to 30-fold reduction in the level of Adr1p . Mutating the strong cAPK phosphorylation site at serine 230 did not suppress this effect . Analysis of ADR1 mRNA levels and ADR1-lacZ expression suggested that decreased ADR1 transcription was responsible for the reduced protein level . In contrast to the ADH2 promoter, however, deletion analysis suggested that cAPK does not act through a discrete DNA element in the ADR1 promoter . The amount of Adr1p found in bcy1 mutant cells should have been sufficient to support 23% of the wild-type level of ADH2 expression . Since no ADH2 expression was detectable in bcy1 mutant cells, cAPK must also act by other mechanisms . Overexpression of Adr1p only partially restored ADH2 expression, indicating that some of these mechanisms may impinge upon events at or subsequent to the ADR1-dependent step in ADH2 transcriptional activation.

Mol Cell Biol, 1997 Mar, 17(3), 1346 - 53
Lck regulates Vav activation of members of the Rho family of GTPases; Han J et al.; Vav is a member of a family of oncogene proteins that share an approximately 250-amino-acid motif called a Dbl homology domain . Paradoxically, Dbl itself and other proteins containing a Dbl domain catalyze GTP-GDP exchange for Rho family proteins, whereas Vav has been reported to catalyze GTP-GDP exchange for Ras proteins . We present Saccharomyces cerevisiae genetic data, in vitro biochemical data, and animal cell biological data indicating that Vav is a guanine nucleotide exchange factor for Rho-related proteins, but in similar genetic and biochemical experiments we fail to find evidence that Vav is a guanine nucleotide exchange factor for Ras . Further, we present data indicating that the Lck kinase activates the guanine nucleotide exchange factor and transforming activity of Vav.

Mol Cell Biol, 1997 Mar, 17(3), 1298 - 313
Homologous segments in three subunits of the guanine nucleotide exchange factor eIF2B mediate translational regulation by phosphorylation of eIF2; Pavitt GD et al.; eIF2B is a five-subunit guanine nucleotide exchange factor that is negatively regulated by phosphorylation of the alpha subunit of its substrate, eIF2, leading to inhibition of translation initiation . To analyze this regulatory mechanism, we have characterized 29 novel mutations in the homologous eIF2B subunits encoded by GCD2, GCD7, and GCN3 that reduce or abolish inhibition of eIF2B activity by eIF2 phosphorylated on its alpha subunit {eIF2(alphaP)} . Most, if not all, of the mutations decrease sensitivity to eIF2(alphaP) without excluding GCN3, the nonessential subunit, from eIF2B; thus, all three proteins are critical for regulation of eIF2B by eIF2(alphaP) . The mutations are clustered at both ends of the homologous region of each subunit, within two segments each of approximately 70 amino acids in length . Several mutations alter residues at equivalent positions in two or all three subunits . These results imply that structurally similar segments in GCD2, GCD7, and GCN3 perform related functions in eIF2B regulation . We propose that these segments form a single domain in eIF2B that makes multiple contacts with the alpha subunit of eIF2, around the phosphorylation site, allowing eIF2B to detect and respond to phosphoserine at residue 51 . Most of the eIF2 is phosphorylated in certain mutants, suggesting that these substitutions allow eIF2B to accept phosphorylated eIF2 as a substrate for nucleotide exchange.

Mol Cell Biol, 1997 Mar, 17(3), 1264 - 73
Molecular and biochemical characterization of xrs mutants defective in Ku80; Singleton BK et al.; The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensively studied xrs mutants, has recently been identified as Ku80, a subunit of the Ku protein and a component of DNA-dependent protein kinase (DNA-PK) . Several group 5 mutants, including xrs-5 and -6, lack double-stranded DNA end-binding and DNA-PK activities . In this study, we examined additional xrs mutants at the molecular and biochemical levels . All mutants examined have low or undetectable levels of Ku70 and Ku80 protein, end-binding, and DNA-PK activities . Only one mutant, xrs-6, has Ku80 transcript levels detectable by Northern hybridization, but Ku80 mRNA was detectable by reverse transcription-PCR in most other mutants . Two mutants, xrs-4 and -6, have altered Ku80 transcripts resulting from mutational changes in the genomic Ku80 sequence affecting RNA splicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression . Neither of these two mutants has detectable wild-type Ku80 transcript . Since the mutation in both xrs-4 and xrs-6 cells results in severely truncated Ku80 protein, both are likely candidates to be null mutants . Azacytidine-induced revertants of xrs-4 and -6 carried both wild-type and mutant transcripts . The results with these revertants strongly support our model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation . Site-directed mutagenesis studies indicate that previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities.

Mol Cell Biol, 1997 Mar, 17(3), 1212 - 23
A family of cyclin-like proteins that interact with the Pho85 cyclin-dependent kinase; Measday V et al.; In budding yeast, entry into the mitotic cell cycle, or Start, requires the Cdc28 cyclin-dependent kinase (Cdk) and one of its three associated G1 cyclins, Cln1, Cln2, or Cln3 . In addition, two other G1 cyclins, Pcl1 and Pcl2, associate with a second Cdk, Pho85, to contribute to Start . Although Pho85 is not essential for viability, Pcl1,2-Pho85 kinase complexes become essential for Start in the absence of Cln1,2-Cdc28 kinases . In addition, Pho85 interacts with a third cyclin, Pho80, to regulate acid phosphatase gene expression . Other cellular roles for Pho85 cyclin-Cdk complexes are suggested by the multiple phenotypes associated with deletion of PHO85, in addition to Start defects and deregulated acid phosphatase gene expression . Strains with pho80, pcl1, and pcl2 deletions show only a subset of the pho85 mutant phenotypes, suggesting the existence of additional Pho85 cyclins (Pcls) . We used two-hybrid screening and database searching to identify seven additional cyclin-related genes that may interact with Pho85 . We found that all of the new genes encode proteins that interacted with Pho85 in an affinity chromatography assay . One of these genes, CLG1, was previously suggested to encode a cyclin, based on the protein's sequence homology to Pcl1 and Pcl2 . We have named the other genes PCL5, PCL6, PCL7, PCL8, PCL9, and PCL10 . On the basis of sequence similarities, the PCLs can be divided into two subfamilies: the Pcl1,2-like subfamily and the Pho80-like subfamily . We found that deletion of members of the Pcl1,2 class of genes resulted in pronounced morphological abnormalities . In addition, we found that expression of one member of the Pcl1,2 subfamily, PCL9, is cell cycle regulated and is decreased in cells arrested in G1 by pheromone treatment . Our studies suggest that Pho85 associates with multiple cyclins and that subsets of cyclins may direct Pho85 to perform distinct roles in cell growth and division.

Mol Cell Biol, 1997 Mar, 17(3), 1160 - 9
Cdc73p and Paf1p are found in a novel RNA polymerase II-containing complex distinct from the Srbp-containing holoenzyme; Shi X et al.; The products of the yeast CDC73 and PAF1 genes were originally identified as RNA polymerase II-associated proteins . Paf1p is a nuclear protein important for cell growth and transcriptional regulation of a subset of yeast genes . In this study we demonstrate that the product of CDC73 is a nuclear protein that interacts directly with purified RNA polymerase II in vitro . Deletion of CDC73 confers a temperature-sensitive phenotype . Combination of the cdc73 mutation with the more severe paf1 mutation does not result in an enhanced phenotype, indicating that the two proteins may function in the same cellular processes . To determine the relationship between Cdc73p and Paf1p and the recently described holoenzyme form of RNA polymerase II, we created yeast strains containing glutathione S-transferase (GST)-tagged forms of CDC73, PAF1, and TFG2 functionally replacing the chromosomal copies of the genes . Isolation of GST-tagged Cdc73p and Paf1p complexes has revealed a unique form of RNA polymerase II that contains both Cdc73p and Paf1p but lacks the Srbps found in the holoenzyme . The Cdc73p-Paf1p-RNA polymerase II-containing complex also includes Gal11p, and the general initiation factors TFIIB and TFIIF, but lacks TBP, TFIIH, and transcription elongation factor TFIIS as well as the Srbps . The Srbp-containing holoenzyme does not include either Paf1p or Cdc73p, demonstrating that these two forms of RNA polymerase II are distinct . In confirmation of the hypothesis that the two forms coexist in yeast cells, we found that a TFIIF-containing complex isolated via the GST-tagged Tfg2p construct contains both (i) the Srbps and (ii) Cdc73p and Paf1p . The Srbps and Cdc73p-Paf1p therefore appear to define two complexes with partially redundant, essential functions in the yeast cell . Using the technique of differential display, we have identified several genes whose transcripts require Cdc73p and/or Paf1p for normal levels of expression . Our analysis suggests that there are multiple RNA polymerase II-containing complexes involved in the expression of different classes of protein-coding genes.

J Biotechnol, 1997 Feb 28, 53(1), 55 - 66
Continuous cultivation start-up control--an experimental investigation; Moller H et al.; A control strategy to avoid development of synchronous growth in carbohydrate limited Saccharomyces cerevisiae cultivations is proposed and experimentally investigated . The basic idea is to control the metabolic flux through the pathways by manipulating the substrate feed rate to keep the ethanol concentration at a low level . An adaptive and a fixed parameter controller were investigated experimentally . Both controllers were initialized at the target conditions for the continuous cultivation, where the uncontrolled process is known to be marginally stable . The latter fact renders it unfeasible to attempt open loop operation at the critical dilution rate . The adaptive controller turned out to be superior to the fixed parameter controller . The superiority of the adaptive controller is ascribed to its ability to identify the process under varying cell activity . The obtained experimental results demonstrate that the desired operating point is reproducibly obtainable . However, after prolonged operation under different types of disturbances the yeast seemed to adapt towards an increased respiratory activity for the same low level of ethanol in the medium.

Mol Cells, 1997 Feb 28, 7(1), 58 - 63
Cloning of CTP:phosphocholine cytidylyltransferase cDNA from Arabidopsis thaliana; Choi SB et al.; As one of the first steps to elucidate the relationship between the structure and function of CTP:phosphocholine cytidylytransferase (EC 2.7.7.15) in plants, the cytidylyltransferase cDNA of Arabidopsis thaliana was cloned and characterized . The A . thaliana cytidylyltransferase cDNA is 1447 bp long and contains an open reading frame of 993 bp coding for a protein of 331 amino acids . The deduced structure of the enzyme was composed of three main regions; the catalytic domain in the N-terminal half, the hydrophilic C-terminal region and the amphipathic domain in the middle . The catalytic domain region was relatively well conserved among different organisms, showing 76 and 72% homology with the rat and yeast protein sequences, respectively . The hydropathy profile revealed that the C-terminal non-catalytic portion of the protein was very hydrophilic, highly enriched in negatively charged aspartic acid and glutamic acid residues . In the region between the catalytic domain and the C-terminal region, there was an amphipathic alpha-helical domain, which was believed to bind the membrane surface in the active formation . Unlike animal counterparts, there was only one potential site of phosphorylation by protein kinase C and none by Ca2+/calmodulin protein kinase II in the C-terminal region . The identity of cytidylyltransferase cDNA was verified by successful transformation of a yeast mutant defective in the enzyme activity, using an expression vector inserted with the A . thaliana cytidylyltransferase cDNA . This was further confirmed by in vivo analysis of the enzyme reaction product after labeling the yeast transformants with radioactive phosphocholine . Southern analysis indicated the presence of a single copy of the citidylyltransferase gene in A . thaliana.

Mol Cells, 1997 Feb 28, 7(1), 45 - 51
Isolation and characterization of a rice MADS box gene belonging to the AGL2 gene family; Kang HG et al.; The MADS box genes that encode regulatory proteins play important roles in both the formation of flower meristem and the determination of floral organ identity . We have characterized a flower-specific cDNA that belongs to the AGL2 gene family from rice, designated OsMADS5 . The cDNA displays the structure of a typical plant MADS box gene, which consists of a MADS domain, K domain, a short I region between the MADS and K domain, and the C-terminal region downstream of the K domain . The gene was classified as a member of the AGL2 gene family from sequence homology analysis . The OsMADS5 protein is the most similar to OsMADS1 of rice (72% identity) . In spatial and temporal RNA blot analyses, the OsMADS5 gene was expressed preferentially in anthers and weakly in carpels . During flower development, the gene was more highly expressed in the early floral stage than in the late . To study the functions of the gene, the cDNA clone was expressed ectopically using the CaMV 35S promoter in a heterologous tobacco plant system . Transgenic plants of OsMADS5 exhibited the phenotype of weak dwarfism and early flowering . These results indicate that OsMADS5 is structurally related to the AGL2 family and may be involved in controlling flowering time.

Gene, 1997 Feb 28, 186(2), 271 - 7
The S3a ribosomal protein gene is identical to the Fte-1 (v-fos transformation effector) gene and the TNF-alpha-induced TU-11 gene, and its transcript level is altered in transformed and tumor cells; Lecomte F et al.; Previous work on mouse x rat hybrid cells (BS series) led to the assignment of a transformation suppressor locus (Sail) to the rat 5q22-q33 region . This gene is not yet identified . From a non-transformed BS hybrid cell line, we isolated a partial cDNA insert (13T), which detects a transcript more abundant in transformed cells than in their non-transformed homologs . Sequence comparisons led us to conclude that 13T is identical to the coding sequences of the ribosomal protein S3a gene (Rps3a), of Fte-1 (v-fos transformation effector gene) and of TU-11, a mouse gene induced by TNF-alpha . Rps3a, Fte-1 and TU-11 are thus one and the same gene . Similarity was also found between this gene and non-mammalian sequences reported to be involved in cell cycling . Like the Rps3a transcript level, the c-Fos transcript level is higher in transformed cells . Rps3a and Fos could thus be effectors of the transformed phenotype.

Gene, 1997 Feb 28, 186(2), 161 - 5
Cloning and characterization of a cDNA encoding bovine mannan-binding protein; Kawai T et al.; To identify the bovine mannan-binding protein (MBP), a search for the cDNA homologue of human MBP was carried out . cDNA clones encoding bovine MBP were isolated from a bovine liver cDNA library using a cDNA fragment encoding a short collagen region, neck domain and carbohydrate recognition domain of human MBP . The cDNA carried an insert of 747 bp encoding a protein of 249 amino acid (aa) residues with a signal peptide of 19 aa . The mannan-binding protein fraction of bovine serum that eluted with 100 mM mannose from a mannan-Sepharose column was analyzed under reducing conditions by SDS-PAGE . The major band of 33 kDa obtained reacted with anti-human MBP rabbit serum . The partial aa sequence of the purified 33-kDa protein was identical to the aa sequence deduced from the obtained cDNA . Results of the passive hemolysis experiment using sheep erythrocytes coated with yeast mannan suggest that this MBP has the ability to activate complement . Northern blot analysis showed a 1.8-kb mRNA that was expressed only in the liver . Based on results of genomic analysis, this bovine MBP is likely to be a homologue of human MBP and to also have homology to rat and mouse MBP-C which are localized in liver cells rather than to rat and mouse MBP-A found in serum . Alignments of bovine collectins show that bovine MBP cannot be included among the other bovine collectins, such as bovine SP-D, conglutinin and CL-43 . Finally, these genomic and biological analyses indicate that the cDNA obtained here encoded a bovine serum MBP.

J Mol Biol, 1997 Feb 28, 266(3), 465 - 78
Synergistic transcriptional-activation by the papillomavirus E2 protein occurs after DNA binding and correlates with a change in chromatin structure; Lefebvre O et al.; The papillomavirus E2 protein only activates transcription strongly when two or more of its binding-sites, each of which bind an E2 dimer, are present upstream of a minimal promoter . Such synergy has been observed both in mammalian and yeast cells . In an attempt to understand the molecular basis of this synergy we carried out genomic footprinting to monitor the binding in vivo of native or mutant E2 proteins to different templates in yeast . We show that in vivo E2 binds to its site even under conditions where it does not activate a reporter gene . Binding occurs at each site independently of the number of sites and even in the absence of the activation domain . In contrast, analysis of the chromatin structure around the E2 binding-site(s) showed that a pronounced change in chromatin structure occurs under conditions in which E2 dimers activate transcription synergistically.

J Biol Chem, 1997 Feb 28, 272(9), 6044 - 50
Relationship between the peptide-sensitive channel and the mitochondrial outer membrane protein translocation machinery; Juin P et al.; The peptide-sensitive channel (PSC), a cationic channel of the mitochondrial outer membrane, is blocked by synthetic mitochondrial presequences and by nonmitochondrial basic peptides such as dynorphin B(1-13) . Both types of peptides are imported into mitochondria . However, the import of dynorphin B(1-13) had to be further characterized since its properties differed from those of the general import pathway used by mitochondrial peptides . Cross-linking experiments with iodinated dynorphin B(1-13) led to the labeling of TOM 40/ISP 42, a component of the protein import machinery of the outer membrane . Accordingly, dynorphin B(1-13) could also be used as a presequence to direct the import of a cytosolic protein into the mitochondria . Pretreatment of intact mitochondria by trypsin removed components capable of discriminating between true mitochondrial presequences and other basic peptides active on the PSC . After proteolysis, both types of peptides appeared to cross the outer membrane through the same pathway . Involvement of the PSC in the translocation complex was shown by immunoprecipitation of the PSC activity by anti-ISP 42 antibodies . Taken together, the present data reinforce the hypothesis that the PSC is the pore responsible for the translocation of protein through the outer membrane.

J Biol Chem, 1997 Feb 28, 272(9), 5765 - 73
Ku selectively transfers between DNA molecules with homologous ends; Bliss TM et al.; Double strand break repair and V(D)J recombination in mammalian cells require the function of the Ku protein complex and the DNA-dependent protein kinase . The DNA-dependent protein kinase is targeted to DNA through its interaction with the Ku protein complex, and thus the specificity of template recognition in the repair and recombination reactions depend on Ku . We have studied Ku binding to DNA using competitive gel shift analysis . We find that Ku bound to one DNA molecule can transfer directly to another DNA molecule when the two DNA molecules have homologous ends containing a minimum of four matched bases . This remarkable reaction can give a false impression of sequence specificity of Ku DNA binding under certain assay conditions . A model is proposed for the DNA binding function of Ku on the basis of these results and the discovery of a novel type of DNA-Ku complex formed at high Ku/DNA ratios is discussed.

J Biol Chem, 1997 Feb 28, 272(9), 5647 - 58
Sequence-specific DNA binding and transcription factor phosphorylation by Ku Autoantigen/DNA-dependent protein kinase . Phosphorylation of Ser-527 of the rat glucocorticoid receptor; Giffin W et al.; NRE1 is a DNA sequence element through which Ku antigen/DNA-dependent protein kinase (DNA-PK) catalytic subunit represses the induction of mouse mammary tumor virus transcription by glucocorticoids . Although Ku is an avid binder of DNA ends and has the ability to translocate along DNA, we report that direct sequence-specific Ku binding occurs with higher affinity (Kd = 0.84 +/- 0.24 nM) than DNA end binding . Comparison of Ku binding to several sequences over which Ku can accumulate revealed two classes of sequence . Sequences with similarity to NRE1 competed efficiently for NRE1 binding . Conversely, sequences lacking similarity to NRE1 competed poorly for Ku and were not recognized in the absence of DNA ends . Phosphorylation of glucocorticoid receptor (GR) fusion proteins by DNA-PK reflected Ku DNA-binding preferences and demonstrated that co-localization of GR with DNA-PK on DNA in cis was critical for efficient phosphorylation . Phosphorylation of the GR fusion protein by DNA-PK mapped to a single site, Ser-527 . This site occurs adjacent the GR nuclear localization sequence between the DNA and ligand binding domains of GR, and thus its phosphorylation, if confirmed, has the potential to affect receptor function in vivo.

J Biol Chem, 1997 Feb 28, 272(9), 5413 - 20
The juxtamembrane, cytosolic region of the epidermal growth factor receptor is involved in association with alpha-subunit of Gs; Sun H et al.; Previously, we have demonstrated that epidermal growth factor (EGF) can stimulate adenylyl cyclase activity via activation of Gs in the heart . Moreover, we have recently shown that Gsalpha is phosphorylated by the EGF receptor protein tyrosine kinase and that the juxtamembrane region of the EGF receptor can stimulate Gs directly . Therefore, employing isolated cardiac membranes, the two-hybrid assay, and in vitro association studies with purified EGF receptor and Gsalpha we have investigated Gsalpha complex formation with the EGF receptor and elucidated the region in the receptor involved in this interaction . In isolated cardiac membranes, immunoprecipitation of EGF receptor was accompanied by co-immunoprecipitation of Gsalpha . In the yeast two-hybrid assay, the cytosolic domain of the EGF receptor and the N-terminal 64 amino acids of this region (Met644-Trp707) associated with Gsalpha . However, interactions of these regions of the EGF receptor with constitutively active Gsalpha were diminished in the two-hybrid assay . Employing purified proteins, our studies demonstrate that the EGF receptor, directly and stoichiometrically, associates with Gsalpha (1 mol of Gsalpha/mol of EGF receptor) . This association was not altered in the presence or absence of ATP and therefore, was independent of tyrosine phosphorylation of either of the proteins . Peptides corresponding to the juxtamembrane region of the receptor decreased association of the EGF receptor with Gsalpha . However, neither the C-terminally truncated EGF receptor (Delta1022-1186) nor a peptide corresponding to residues 985-996 of the receptor altered association with Gsalpha, thus indicating the selectivity of the G protein interaction with the juxtamembrane region . Interestingly, peptides corresponding to N and C termini of Gsalpha did not alter the association of Gsalpha with the EGF receptor . Consistent with the findings from the two-hybrid assay where constitutively active Gsalpha poorly associated with the EGF receptor, in vitro experiments with purified proteins also demonstrated that activation of Gsalpha by guanosine 5'-3-O-(thio)triphosphate decreased the association of G protein with the EGF receptor . Thus we conclude that the juxtamembrane region of the EGF receptor, directly and stoichiometrically, associates with Gsalpha and that upon activation of Gsalpha this association is decreased.

Science, 1997 Feb 28, 275(5304), 1311 - 4
APC-mediated proteolysis of Ase1 and the morphogenesis of the mitotic spindle; Juang YL et al.; The molecular mechanisms that link cell-cycle controls to the mitotic apparatus are poorly understood . A component of the Saccharomyces cerevisiae spindle, Ase1, was observed to undergo cell cycle-specific degradation mediated by the cyclosome, or anaphase promoting complex (APC) . Ase1 was degraded when cells exited from mitosis and entered G1 . Inappropriate expression of stable Ase1 during G1 produced a spindle defect that is sensed by the spindle assembly checkpoint . In addition, loss of ASE1 function destabilized telophase spindles, and expression of a nondegradable Ase1 mutant delayed spindle disassembly . APC-mediated proteolysis therefore appears to regulate both spindle assembly and disassembly.

Exp Cell Res, 1997 Feb 25, 231(1), 198 - 205
Regulation of ribosomal RNA gene transcription during retinoic acid-induced differentiation of mouse teratocarcinoma cells; Datta PK et al.; We have examined the mechanism of regulation of rRNA synthesis in mouse F9 teratocarcinoma cells that were induced to differentiate by retinoic acid and dibutyryl cAMP . Ribosomal RNA (rRNA) synthesis was significantly reduced during differentiation of F9 cells into parietal endoderm cells . Nuclear run-on assay revealed that the rRNA gene transcription rates were reduced in differentiated cells, and this phenomenon could be mimicked by in vitro transcription assay using nuclear extracts prepared from F9 stem and F9 parietal endoderm cells . Analysis of the DNA-binding activities of two RNA polymerase I (pol I) transcription factors E1BF/Ku and UBF revealed decreased affinity for their cognate recognition sequences . Immunoblot analysis showed a marked reduction in the amounts of E1BF/Ku and UBF in the differentiated cells . Analysis of the steady-state RNA levels for the smaller subunit of E1BF/Ku and for UBF in differentiating F9 cells revealed decreased mRNA synthesis and increase in message level for the differentiation-specific marker laminin B1 with progression of the differentiated status of the cells . This study has demonstrated that differentiation of mouse F9 teratocarcinoma cells into parietal endoderm cells leads to diminished rRNA synthesis, which may be mediated by reduced DNA-binding activities and amounts of at least two pol I transcription factors.

J Cell Biol, 1997 Feb 24, 136(4), 803 - 10
Identification of novel vesicles in the cytosol to vacuole protein degradation pathway; Huang PH et al.; The key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae are starved of glucose . FBPase is targeted from the cytosol to the yeast vacuole for degradation when glucose-starved cells are replenished with fresh glucose . Several vid mutants defective in the glucose-induced degradation of FBPase in the vacuole have been isolated . In some vid mutants, FBPase is found in punctate structures in the cytoplasm . When extracts from these cells are fractionated, a substantial amount of FBPase is sedimentable in the high speed pellet, suggesting that FBPase is associated with intracellular structures in these vid mutants . In this paper we investigated whether FBPase association with intracellular structures also existed in wild-type cells . We report the purification of novel FBPase-associated vesicles from wild-type cells to near homogeneity . Kinetic studies indicate that FBPase association with these vesicles is stimulated by glucose and occurs only transiently, suggesting that these vesicles are intermediate in the FBPase degradation pathway . Fractionation analysis demonstrates that these vesicles are distinct from known organelles such as the vacuole, ER, Golgi, mitochondria, peroxisomes, endosomes, COPI, or COPII vesicles . Under EM, these vesicles are 30-40 nm in diam . Proteinase K experiments indicate that the majority of FBPase is sequestered inside the vesicles . We propose that FBPase is imported into these vesicles before entering the vacuole.

Mol Gen Genet, 1997 Feb 20, 253(5), 642 - 8
Molecular characterization of the deep orange (dor) gene of Drosophila melanogaster; Shestopal SA et al.; Mutations of the dor gene of Drosophila melanogaster cause defects in different stages of development . Heterozygotes for lethal or viable dor alleles and the rearrangement T(1;2)dor(var7), which causes position effect variegation of dor, exhibit traits such as rough eyes, reduction of bristles on the thorax and scutellum and wavy wings . The dor gene was mapped to the proximal part of the 2B3-5 band or in the interband between 2B3-5 and 2B6 and localised within an interval of 5 kb on the physical map of the cloned 2B region . The 3.0-3.1 kb dor transcript was detected by Northern hybridization at all stages of development and is expressed in salivary glands of third instar larve . This RNA was not expressed in the dor mutants with insertions in the 5' part of the gene . The sequence of the 3180 bp (dor cDNA predicts a 115.3 kDa protein that contains a cysteine- and histidine-rich zinc finger-like motif CX2CX13CXHX2HX2CX2H at the C-terminus . The protein sequence reveals 23% identity to the Saccharomyces cerevisiae PEP3 protein . The most significant homology (57%) similarity and 32%, identity) between the DOR and PEP3 proteins is observed at the C-termini of the proteins.

Mol Gen Genet, 1997 Feb 20, 253(5), 553 - 61
Dissection of the wheat transcription factor HBP-1a(17) reveals a modular structure for the activation domain; Nakayama T et al.; The wheat bZIP protein HBP-1a(17) is a putative transcription factor regulating histone gene expression . To delineate the functional domain(s) of this factor, we made a series of effector constructs expressing fusion proteins, in which various portions of HBP-1a(17) are fused to the DNA-binding domain of the yeast transcriptional activator GAL4, in plant cells . When the beta-glucuronidase (GUS) reporter gene, driven by the wheat histone H3 core promoter harboring the GAL4-binding sequence, was co-transfected with such effector genes into tobacco protoplasts, several portions of HBP-1a(17) influenced reporter gene expression . The N-terminal one-third of HBP-1a(17), termed the P region (residues 1-118) due to its Pro content, did not activate the reporter gene, in contrast to the corresponding Pro-rich region of Arabidopsis GBF1 (residues 1-110), which functions as an activation domain . When the P region was divided into two, however, both its N-terminal (1-56; termed NP) and C-terminal (58-118; termed PC) halves were able to enhance expression of the reporter gene . When the NP region was further divided into NP(5-30) and NP(30-56), both regions still retained activating ability . These results suggest that the P region of HBP-1a(17) is composed of several modules each having activating function, and modification and/or conformational changes of the P region might influence its function.

Brain Res Dev Brain Res, 1997 Feb 20, 98(2), 197 - 203
Acetyl-CoA carboxylase gene expression in the developing mouse brain . Comparison with other genes involved in lipid biosynthesis; Garbay B et al.; The present study documents the steady-state levels for the mRNAs encoding acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD2) and brain long-chain acyl-CoA synthetase (BLACS) during mouse brain development . It is shown that ACC and FAS mRNA levels are at a maximum 5 days after birth, a time when cell proliferation is intense in the mouse brain, and then decrease steadily to reach 20% of those maximal values at day 20 . The ACC transcript isoforms, which were detected in the central nervous system (CNS), originated from promoter P2 of the ACC gene . They encode ACC enzymes which cannot be phosphorylated at the Ser-1200 locus, thus indicating that brain ACC is highly sensitive to citrate activation . The developmental pattern for the SCD2 mRNA level is different from that of true myelin genes, such as CGT . Indeed, the steady-state levels for SCD2 and CGT in 5-day-old brain represent 85% and 5% of their maximal values, respectively . BLACS expression rose during the developmental period studied, but a slow decrease in the mRNA levels was not observed after postnatal day 20, unlike in "myelin-specific' genes . Therefore, it appears that the expression of the genes involved in fatty acid biosynthesis is independent of the myelinating signal in the mouse CNS.

Biochemistry, 1997 Feb 18, 36(7), 1912 - 7
Internally consistent libraries of fluorogenic substrates demonstrate that Kex2 protease specificity is generated by multiple mechanisms; Rockwell NC et al.; Kex2 protease from the yeast Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases . To clarify understanding of the interactions responsible for substrate recognition in this family of enzymes, we have carried out a systematic examination of Kex2 substrate specificity using internally consistent sets of substrates having substitutions at only one or two positions . We examined Kex2 sequence recognition for residues at P3, P2, and P1 using two types of fluorogenic peptide substrates, peptidyl-methylcoumarinamides and internally quenched substrates in which cleavage occurs at an actual peptide bond . Kinetic analysis of the two sets of substrates gave comparable data on specificity at these three positions . For the best substrate sequences, high catalytic constants (kCM/KM) of (2-5) x 10(7) M-1 s-1 were seen for cleavage of both peptidyl-methylcoumarinamides and peptide bonds . While no evidence for positive interactions with the P3 residue emerged, Kex2 was found to discriminate against at least one residue Asp . at this position . Specificity at P2 was shown to rely primarily on recognition of a positive charge, although steric constraints on the P2 side chain were also apparent . Kex2 was demonstrated to be exquisitely selective for Arg at P1 . Substitutions with similar charge (Lys, ornithine) or similar hydrogen-bonding capability (citrulline) do not confer efficient catalysis . Comparison of otherwise identical substrates having either Arg or citrulline at P1 showed that the positive charge of the Arg guanidinium group stabilizes the transition state by approximately 6.8 kcal/mol.

Biochemistry, 1997 Feb 18, 36(7), 1740 - 7
Double-reciprocal crossover mediated by FLP-recombinase: a concept and an assay; Seibler J et al.; FLP recombinase induces a double-reciprocal crossover event between sets of different FLP recognition target (FRT) sites . Therefore, if these sites flank an expression cassette at a given genomic locus, it can be exchanged for another cassette that has been constructed in the analogous was {Schlake & Bode (1994) Biochemistry 33, 12746-12751} . Here we demonstrate that an integrated expression cassette, flanked by a wild type and a mutated site, remains completely stable in the presence of constitutive FLP activity, obviating the need for a timing of this parameter . Therefore the only variable left for optimization is the initial concentration of the exchange plasmid . Since the exchange plasmid lacks a promoter, random integration is not expected to confer resistance to the selection marker, the expression of which requires the acquisition of the SV40 promoter provided at the predetermined integration site . Due to the presence of a luciferase reporter in a specific bicistronic expression cassette, recombination generates bioluminescence upon recombination, indicating the extent of the exchange reaction . This principle is utilized to compare the potential of various cell lines to support the exchange reaction and to adjust the optimum parameters.

FEBS Lett, 1997 Feb 17, 403(2), 186 - 90
Gcn5p is involved in the acetylation of histone H3 in nucleosomes; Ruiz-Garcia AB et al.; Enzymatic extracts from a gcn5 mutant and wild-type strains of Saccharomyces cerevisiae were chromatographically fractionated and the histone acetyltransferase activities compared . When free histones were used as substrate, extracts from wild-type cells showed two peaks of activity on histone H3 but extracts from gcn5 mutant cells showed only one . With nucleosomes as substrate, the histone acetyltransferase activities present in extracts from the gcn5 mutant strain were not able to modify H3 whereas wild-type cell extracts acetylated intensely this histone . The activity that acetylated nucleosome-bound H3 behaved as a 170-kDa complex . We suggest that Gcn5p represents a catalytic subunit within a multiprotein complex containing proteins that confer on it the ability to acetylate H3 in nucleosomes.

Eur J Biochem, 1997 Feb 15, 244(1), 128 - 33
Characterization of the active site of Schwanniomyces occidentalis glucoamylase by in vitro mutagenesis; Hulseweh B et al.; Site-directed mutagenesis was performed to define the active site of the Schwanniomyces occidentalis glucoamylase . The mutated GAM1 genes were expressed in Saccharomyces cerevisiae, and enzymatic and growth properties of the transformants were determined . Mutants were transcribed and translated similar to the wild-type glucoamylase . Therefore, all effects on enzymatic activity could be referred to single amino acid substitutions . Asp470 was shown to be essential for the enzyme activity . Replacement of Asp470 by glycine led to a complete loss of activity . We suppose that Asp470 serves as a general acid-base and stabilizes the formation of the intermediate carbenium ion . Substitution of Trp468 by alanine affected predominantly the alpha-1,6 activity and not the alpha-1,4 activity of the enzyme . The exchange impaired substrate binding as well as enzymatic catalysis . An influence of amino acid 474 on the substrate specificity could not be demonstrated . Exchanges at position 474 exhibited K(m) and Vmax values similar to wild-type glucoamylase.

Nucleic Acids Res, 1997 Feb 15, 25(4), 888 - 96
Functional analysis of the polypyrimidine tract in pre-mRNA splicing; Coolidge CJ et al.; The polypyrimidine tract is one of the important cis-acting sequence elements directing intron removal in pre-mRNA splicing . Progressive deletions of the polypyrimidine tract have been found to abolish correct lariat formation, spliceosome assembly and splicing . In addition, the polypyrimidine tract can alter 3'-splice site selection by promoting alternative branch site selection . However, there appears to be great flexibility in the specific sequence of a given tract . Not only the optimal composition of the polypyrimidine tract, but also the role of the tract in introns with no apparent polypyrimidine tracts or where changes in the tract are apparently harmless are uncertain . Accordingly, we have designed a series of cis-competition splicing constructs to test the functional competitive efficiency of a variety of systematically mutated polypyrimidine tracts . An RT/PCR assay was used to detect spliced product formation as a result of differential branch point selection dependent on direct competition between two opposing polypyrimidine tracts . We found that pyrimidine tracts containing 11 continuous uridines are the strongest pyrimidine tracts . In such cases, the position of the uridine stretch between the branch point and 3'-splice site AG is unimportant . In contrast, decreasing the continuous uridine stretch to five or six residues requires that the tract be located immediately adjacent to the AG for optimal competitive efficiency . The block to splicing with decreasing polypyrimidine tract strength is primarily prior to the first step of splicing . While lengthy continuous uridine tracts are the most competitive, tracts with decreased numbers of consecutive uridines and even tracts with alternating purine/pyrimidine residues can still function to promote branch point selection, but are far less effective competitors in 3'-splice site selection assays.

J Biol Chem, 1997 Feb 14, 272(7), 4613 - 22
Genomic footprinting of Mig1p in the MAL62 promoter . Binding is dependent upon carbon source and competitive with the Mal63p activator; Wang J et al.; Mig1p inhibits gene expression in glucose by binding the Cyc8p (Ssn6p)-Tup1p repressor to the promoter of glucose-repressible genes . While the binding properties of Mig1p have been studied in vitro and the ability of Mig1p-Cyc8p (Ssn6p)-Tup1p to repress has been studied in vivo, no experiments have measured the effect of a carbon source on the in vivo binding of Mig1p or the effect of bound MIg1p on activator occupancy of the upstream activation sequence (UAS) . To obtain this information, we used genomic footprinting to investigate glucose repression of MAL62, a gene that is also regulated by the Mal63p activator . These experiments show that two interrelated mechanisms are involved in the glucose repression of MAL62: 1) competition between the Mal63p activator and Mig1p for DNA binding and 2) modulation of Mig1p binding by the carbon source . Mig1p affects basal MAL62 expression in the absence of Mal63p by binding to a site in the MAL62 promoter and affects Mal63p-dependent synthesis by also inhibiting the access of Mal63p to site 1 in the UASMAL . The binding of Mig1p is increased in glucose and decreased in nonrepressing sugars, but the increased binding in glucose is not due to an increase in the levels of Mig1p.

J Biol Chem, 1997 Feb 14, 272(7), 4451 - 7
Two murine homologs of the Drosophila single-minded protein that interact with the mouse aryl hydrocarbon receptor nuclear translocator protein; Probst MR et al.; Drosophila single-minded, which acts as a positive master gene regulator in central nervous system midline formation in Drosophila, its two mouse homologs SIM1 and SIM2, and the mammalian aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins are members of the basic-helix-loop-helix.PAS family of transcription factors . In the yeast two-hybrid system, we demonstrate strong constitutive interaction of ARNT with SIM1 and SIM2 and fully ligand-dependent interaction of ARNT with AHR . Both the helix-loop-helix and the PAS regions of SIM1 and of ARNT are required for efficient heterodimerization . SIM1 and SIM2 do not form homodimers, and they do not interact with AHR . We also failed to detect homodimerization of ARNT . The interaction of ARNT with SIM1 was confirmed with in vitro synthesized proteins . Like AHR, in vitro synthesized SIM1 associates with the 90-kDa heat shock protein . SIM1 inhibits binding of the AHR.ARNT dimer to the xenobiotic response element in vitro . Introduction of SIM1 into hepatoma cells inhibits transcriptional transactivation by the endogenous AHR.ARNT dimer . The mouse SIM1 . ARNT dimer binds only weakly to a proposed DNA target for the Drosophila SIM.ARNT dimer . In adult mice mRNA for SIM1 was expressed in lung, skeletal muscle, and kidney, whereas the mRNA for SIM2 was found in the latter two . ARNT is also expressed in these organs . Thus mouse SIM1 and SIM2 are novel heterodimerization partners for ARNT in vitro, and they may function both as positive and negative transcriptional regulators in vivo, during embryogenesis and in the adult organism.

J Biol Chem, 1997 Feb 14, 272(7), 4436 - 43
Oncogenic point mutations induce altered conformation, redox sensitivity, and DNA binding in the minimal DNA binding domain of avian myeloblastosis virus v-Myb; Brendeford EM et al.; c-Myb is the founder member of a class of transcription factors with tryptophan-rich repeats responsible for DNA binding . Activated oncogenic forms of Myb are encoded by the avian retroviruses, avian myeloblastosis virus (AMV) and E26 . AMV v-Myb encodes a truncated protein with 11 point mutations relative to c-Myb . The mutations in the DNA binding domain (DBD) were reported to impose distinct phenotypes of differentiation on transformed myeloid cells (Introna, M., Golay, J., Frampton, J., Nakano, T., Ness, S . A., and Graf, T . (1990) Cell 63, 1287-1297) . The molecular mechanism operating has remained elusive since no change in sequence specificity has been found . We introduced AMV-specific point mutations in the minimal DBD of chicken c-Myb and studied their effect on structure and function of the purified protein . Fluorescence emission spectra and fluorescence quenching experiments showed that the AMV-specific point mutations had a significant effect on the conformation of the DBD, giving rise to a more compact structure, a change that was accompanied by a reduced sensitivity toward cysteine-specific alkylation and oxidation . The DNA binding properties were also altered by the AMV-specific point mutations, leading to protein-DNA complexes with highly reduced stability . This reduction in stability was, however, more severe with certain subtypes of binding sequences than with others . This differential behavior was also observed in an in vivo model system where DBD-VP16 fusions were coexpressed with various reporters . These findings imply that different subsets of Myb-responsive promoters may react differentially toward the AMV-specific mutations, a phenomenon that could contribute to the altered patterns of gene expression induced by the AMV v-Myb relative to wild type c-Myb.

J Biol Chem, 1997 Feb 14, 272(7), 4149 - 56
Three amino acid substitutions selectively disrupt the activation but not the repression function of the glucocorticoid receptor N terminus; Iniguez-Lluhi JA et al.; A 210-amino acid region, termed enh2, near the N terminus of the rat glucocorticoid receptor, is necessary for both transcriptional activation and repression . The mechanism(s) of transcriptional regulation conferred by this region, however, are poorly understood . We screened in Saccharomyces cerevisiae a library of random mutants in the enh2 region of a constitutive glucocorticoid receptor derivative and isolated a series of multiply substituted receptors that are specifically defective in transcriptional activation . Although many substitutions in this area were tolerated, three amino acid substitutions (E219K, F220L, and W234R) within a 16-amino acid region were sufficient to disrupt the enh2 transcriptional activation function both in yeast and in mammalian cells . Although this region is rich in acidic residues, the conserved tryptophan at position 234 appears to be a more critical feature for enh2 activity; hydrophobic but not charged residues were tolerated at this position . Notably, the mutants uncoupled the activation and repression functions of enh2, as the activation defective isolates remained competent for repression of AP-1 at the composite response element plfG.

J Biol Chem, 1997 Feb 14, 272(7), 4141 - 8
Comparative activity of ADP-ribosylation factor family members in the early steps of coated vesicle formation on rat liver Golgi membranes; Liang JO et al.; We have compared the abilities of mammalian ADP-ribosylation factors (ARFs) 1, 5, and 6 and Saccharomyces cerevisiae ARF2 to serve as substrates for the rat liver Golgi membrane guanine nucleotide exchange factor and to initiate the formation of clathrin- and coatomer protein (COP) I-coated vesicles on these membranes . While Golgi membranes stimulated the exchange of GTPgammaS for GDP on all of the ARFs tested, mammalian ARF1 was the best substrate, with an apparent Km of 5 microM . In all cases myristoylation of ARF was required for stimulation . Agents that inhibit the Golgi membrane guanine nucleotide exchange factor (the fungal metabolite brefeldin A and trypsin treatment) selectively inhibited the guanine nucleotide exchange on mammalian ARF1 . Taken together, these data indicate that of the ARFs tested, only mammalian ARF1 is activated efficiently by the Golgi guanine nucleotide exchange factor . The other ARFs are activated mainly by another mechanism, possibly phospholipid-mediated . Once activated, all of the membrane-associated, myristoylated ARFs promoted the recruitment of coatomer to about the same extent . Mammalian ARFs 1 and 5 were the most effective in promoting the recruitment of the AP-1 adaptor complex, whereas yeast ARF2 was the least active . These data indicate that the specificity for ARF action on the Golgi membranes is primarily determined by the Golgi guanine nucleotide exchange factor, which has a strong preference for myristoylated mammalian ARF1.

J Biol Chem, 1997 Feb 14, 272(7), 4021 - 6
Sp3 is a bifunctional transcription regulator with modular independent activation and repression domains; Majello B et al.; Sp3 is a member of the Sp family of transcription factors and binds to DNA with affinity and specificity comparable to that of Sp1 . We demonstrate that Sp3 is a bifunctional transcription factor that can both activate and repress transcription . Gene fusion experiments in mammalian cells demonstrate that the Sp3 activation potential is distributed over an extensive glutamine-rich N-terminal region, whereas the repressor activity has been mapped in a 72-amino acid region located at the 5' of the zinc finger DNA-binding domain . We demonstrated that the repression activity is strictly dependent on the context of the DNA-binding sites bound by Sp3 . We found that Sp3 represses transcription of promoters bearing multiple GAL4 DNA-binding sites, whereas it activates isogenic reporters containing a single GAL4-binding site . Transfection experiments in Drosophila cells that lack endogenous Sp activity demonstrated that Sp3 does not possess an active repression domain that can function in insect cells, rather it is a weak transcriptional activator of the c-myc promoter . Our results strongly suggest that Sp3 is a dual-function regulator whose activity is dependent upon both the promoter and the cellular context.

Science, 1997 Feb 14, 275(5302), 980 - 3
Structural and functional conservation of the Caenorhabditis elegans timing gene clk-1; Ewbank JJ et al.; Mutations in the Caenorhabditis elegans gene clk-1 affect biological timing and extend longevity . The gene clk-1 was identified, and the cloned gene complemented the clk-1 phenotypes and restored normal longevity . The CLK-1 protein was found to be conserved among eukaryotes, including humans, and structurally similar to the yeast metabolic regulator Cat5p (also called Coq7p) . These proteins contain a tandem duplication of a core 82-residue domain . clk-1 complemented the phenotype of cat5/coq7 null mutants, demonstrating that clk-1 and CAT5/COQ7 share biochemical function and that clk-1 acts at the level of cellular physiology.

Oncogene, 1997 Feb 13, 14(6), 653 - 9
A novel human SPS1/STE20 homologue, KHS, activates Jun N-terminal kinase; Tung RM et al.; STE20-homologous proteins have been implicated in mammalian MAP kinase pathways as important transducers of signals from p21 family GTPases . We have cloned a novel STE20 family member, which we call KHS for kinase homologous to SPS1/STE20, that encodes a kinase of 95 kD which is expressed in a variety of tissues . Transiently expressed fusion protein GST-KHS exhibits phosphotransferase activity toward a panel of test substrates, including myelin basic protein (MBP), which is phosphorylated by all known STE20 homologues . KHS is most closely related to another human STE20, GC kinase (74% similar in the catalytic domain), which has recently been placed upstream of the stress-activated MAP kinases (SAPKs/JNKs) . KHS also activates JNK in transient coexpression experiments, suggesting a role for KHS in the stress response of fibroblasts . Characterization and comparison of the regulation of these two kinases will be important in elucidating MAP kinase signalling cascades.

Nature, 1997 Feb 13, 385(6617), 653 - 6
Interaction between the C . elegans cell-death regulators CED-9 and CED-4; Spector MS et al.; Programmed cell death (apoptosis) is an evolutionarily conserved process used by multicellular organisms to eliminate cells that are not needed or are potentially detrimental to the organism . Members of the Bcl-2 family of mammalian proteins are intimately involved in the regulation of apoptosis, but, their precise mechanism of action remains unresolved . In Caenorhabditis elegans, the Bcl-2 homologue CED-9 prevents cell death by antagonizing the death-promoting activities of CED-3, a member of the Caspase family of death proteases, and of CED-4, a protein with no known mammalian homologue . Here we show that CED-9 interacts physically with CED-4 . Mutations that reduce or eliminate CED-9 activity also disrupt its ability to bind CED-4, suggesting that this interaction is important for CED-9 function . Thus, CED-9 might control C . elegans cell death by binding to and regulating CED-4 activity . We propose that mammalian Bcl-2 family members might control apoptosis in a similar way through interaction and regulation of CED-4 homologues or analogues.

J Biol Chem, 1997 Feb 7, 272(6), 3845 - 51
Degradation of E2A proteins through a ubiquitin-conjugating enzyme, UbcE2A; Kho CJ et al.; The helix-loop-helix E2A proteins (E12 and E47) govern cellular growth and differentiation . To identify binding partners that regulate the function of these ubiquitous transcription factors, we screened for proteins that interacted with the C terminus of E12 by the yeast interaction trap . UbcE2A, a rat enzyme that is highly homologous to and functionally complements the yeast ubiquitin-conjugating enzyme UBC9, was identified and cloned . UbcE2A appears to be an E2A-selective ubiquitin-conjugating enzyme because it interacts specifically with a 54-amino acid region in E47-(477-530) distinct from the helix-loop-helix domain . In contrast, most of the UbcE2A protein is required for interaction with an E2A protein . The E2A proteins appear to be degraded by the ubiquitin-proteasome pathway because the E12 half-life of 60 min is extended by the proteasome inhibitor MG132, and E12 is multi-ubiquitinated in vivo . Finally, antisense UbcE2A reduces E12 degradation . By participating in the degradation of the E2A proteins, UbcE2A may regulate cell growth and differentiation.

J Biol Chem, 1997 Feb 7, 272(6), 3766 - 72
Organization and expression of the mouse MTH1 gene for preventing transversion mutation; Igarashi H et al.; An enzyme, 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase (8-oxo-dGTPase), is present in various organisms and plays an important role in the control of spontaneous mutagenesis . The enzyme hydrolyzes 8-oxo-dGTP, an oxidized form of dGTP, to 8-oxo-dGMP, thereby preventing the occurrence of A:T to C:G transversion, caused by misincorporation . We isolated the mouse genomic sequence encoding the enzyme and elucidated its structure . The gene, named MTH1 for mutT homologue 1, is composed of at least five exons and spans approximately 9 kilobase pairs . A genomic region containing the pseudogene was also isolated . The promoter region for the gene is GC-rich, contains many AP-1 and AP-2 recognition sequences, and lacks a typical TATA box . Primer extension and S1 mapping analyses revealed the existence of multiple transcription initiation sites, among which a major site was defined as +1 . The putative promoter region was placed upstream of the chloramphenicol acetyltransferase reporter gene, and control of expression of the gene was examined by introducing the construct into mouse NIH 3T3 cells . Deletion analysis indicated that a sequence from -321 to +9 carries the basic promoter activity while an adjacent region, spanning from +352 to +525 stimulates the frequency of transcription.

J Biol Chem, 1997 Feb 7, 272(6), 3280 - 8
Structural features of alkylphenolic chemicals associated with estrogenic activity; Routledge EJ et al.; The ability of certain man-made chemicals to mimic the effects of natural steroid hormones and their potential to disrupt the delicate balance of the endocrine system in animals are of increasing concern . The growing list of reported hormone-mimics includes the alkylphenolic (AP) compounds, a small number of which have been reported to be weakly estrogenic . In their most basic form, APs are composed of an alkyl group, which can vary in size, branching, and position, joined to a phenolic ring . The aim of this project was to identify the important structural features responsible for the estrogenic activity of AP chemicals . This was achieved by incubating APs with different structural features in a medium containing a previously described estrogen-inducible strain of yeast (Saccharomyces cerevisiae) expressing the human estrogen receptor and comparing their activity spectrophotometrically by the resulting color change of the medium . The results were compared to the effects of the main natural estrogen 17beta-estradiol . The data indicate that both the position (para > meta > ortho) and branching (tertiary > secondary = normal) of the alkyl group affect estrogenicity . Optimal estrogenic activity requires a single tertiary branched alkyl group composed of between 6 and 8 carbons located at the para position on an otherwise unhindered phenol ring . The results are discussed in relation to the purity and composition of the chemicals tested.

Nature, 1997 Feb 6, 385(6616), 552 - 5
The nuclear hormone receptor Ftz-F1 is a cofactor for the Drosophila homeodomain protein Ftz; Yu Y et al.; Homeobox genes specify cell fate and positional identity in embryos throughout the animal kingdom . Paradoxically, although each has a specific function in vivo, the in vitro DNA-binding specificities of homeodomain proteins are overlapping and relatively weak . A current model is that homeodomain proteins interact with cofactors that increase specificity in vivo . Here we use a native binding site for the homeodomain protein Fushi tarazu (Ftz) to isolate Ftz-F1, a protein of the nuclear hormone-receptor superfamily and a new Ftz cofactor . Ftz and Ftz-F1 are present in a complex in Drosophila embryos . Ftz-F1 facilitates the binding of Ftz to DNA, allowing interactions with weak-affinity sites at concentrations of Ftz that alone bind only high-affinity sites . Embryos lacking Ftz-F1 display ftz-like pair-rule cuticular defects . This phenotype is a result of abnormal ftz function because it is expressed but fails to activate downstream target genes . Cooperative interaction between homeodomain proteins and cofactors of different classes may serve as a general mechanism to increase HOX protein specificity and to broaden the range of target sites they regulate.

Nature, 1997 Feb 6, 385(6616), 540 - 4
MAP3K-related kinase involved in NF-kappaB induction by TNF, CD95 and IL-1; Malinin NL et al.; Several members of the tumour-necrosis/nerve-growth factor (TNF/NGF) receptor family activate the transcription factor NF-kappaB through a common adaptor protein, Traf2 (refs 1-5), whereas the interleukin 1 type-I receptor activates NF-kappaB independently of Traf2 (ref . 4) . We have now cloned a new protein kinase, NIK, which binds to Traf2 and stimulates NF-kappaB activity . This kinase shares sequence similarity with several MAPKK kinases . Expression in cells of kinase-deficient NIK mutants fails to stimulate NF-kappaB and blocks its induction by TNF, by either of the two TNF receptors or by the receptor CD95 (Fas/Apo-1), and by TRADD, RIP and MORT1/FADD, which are adaptor proteins that bind to these receptors . It also blocked NF-kappaB induction by interleukin-1 . Our findings indicate that NIK participates in an NF-kappaB-inducing signalling cascade common to receptors of the TNF/NGF family and to the interleukin-1 type-I receptor.

Biochemistry, 1997 Feb 4, 36(5), 1052 - 64
Functional in vivo interaction between the amino-terminal, transactivation domain and the ligand binding domain of the androgen receptor; Doesburg P et al.; The ligand binding domain (LBD) and the amino-terminal, transactivation domain (TAD) of the androgen receptor (AR) were separately linked to the GAL4 DNA binding domain (DBD) and to the GAL4(TAD) . Resulting constructs were tested in the yeast two-hybrid system for protein-protein interactions . In the presence of androgen {methyltrienolone (R1881) or dihydrotestosterone (DHT)} a transcriptionally active complex was formed, reflecting an association between the AR(LBD) and the AR(TAD) . No interactions were found in the presence of low-affinity ligands like estradiol (E2), promegestone (R5020), or progesterone (Pg) . Use of the Thr-868-Ala mutated AR(LBD) in the assay resulted not only in a clear AR TAD-LBD interaction in the presence of R1881 and DHT but also in the presence of E2, Pg, and R5020, corresponding to the alteration in ligand specificity induced by the mutation . Coexpression of the fusion protein Gal4(DBD)AR(LBD) and the separate AR(TAD) also gave rise to the formation of a transcriptionally active complex . No interactions were found between two AR LBDs at the low-expression level of the two components . However, LBD-LBD interaction was detectable by application of a high-expression vector for GAL4(TAD)AR(LBD), albeit at high ligand concentrations . To substantiate the observation of the AR LBD-TAD interaction, CHO cells were cotransfected with expression plasmids for a truncated AR, which lacks the TAD {AR(DBD)(LBD)}, and for the separate AR(TAD) . This resulted in stimulation of a MMTV-LUC reporter gene in the presence of R1881 but not in the absence of hormone . This finding indicates that, like in the yeast system, in mammalian cells, TAD-LBD interactions are of importance for AR activation . In the mammalian system, a maximal AR TAD-LBD interaction was obtained at approximately 10-fold higher ligand concentrations than required for full-length AR activation . In the presence of low-affinity ligands, the AR TAD-LBD interaction as measured by transcriptional activation was considerably weaker than the activity of the full-length AR . From the present results a concept of hormone-dependent AR activation is proposed, which requires a functional, direct or indirect intramolecular interaction between the TAD and the LBD.

Biochemistry, 1997 Feb 4, 36(5), 995 - 1002
Crosslinking kinetics of the human transglutaminase, factor XIII{A2}, acting on fibrin gels and gamma-chain peptides; Lewis KB et al.; Factor XIII is the terminal enzyme of the coagulation cascade which serves to rapidly crosslink the adjacent gamma-chain C-termini of fibrin clots . In vivo, this process is initiated by the proteolytic action of thrombin which simultaneously converts both soluble fibrinogen to fibrin and activates zymogen FXIII; fibrin then spontaneously polymerizes to form a gel which activated FXIII stabilizes through crosslinking . Due to the kinetic complexity and the difficulty of investigating gel phase reactions, methods employing pre-activation of recombinant human Factor XIII (rFXIII{A'2}) were developed to effectively decouple these reactions . By utilizing these methods, the kinetic parameters of gamma-chain crosslinking in fibrin gels could be determined by both initial rate and integrated rate techniques under physiologically relevant conditions . The crosslinking of the gamma-chain of fibrin gels could be described by apparent Michaelis kinetics with K(m)(app) = 6.2 microM, kcat = 1872 min-1, and Ksp = 302 min-1 microM-1 for a fibrin gamma-chain monomer of M(r) = 170000 Da . In contrast, both the crosslinking rates of alpha-chains within fibrin gels (Ksp = 0.38 min-1 microM-1: Bishop et al . (1993)) and the crosslinking of a soluble synthetic peptide containing the unique gamma-chain fibrin crosslinking site (Ksp = 0.030 min-1 microM-1) could not be shown to saturate and gave apparent first-order rates with respect to rFXIII{A'2} . These observations coupled with the large differences in the turnover rates (approximately 10(4)) suggest two likely mechanisms for FXIII{A'2}-substrate interactions: (1) random (or independent) binding of non- or weakly interacting substrate pairs imposes a high entropic barrier (i . e., delta Gbinding) to the formation of a productive catalytic complex, e.g., for soluble gamma-chain peptides and the flexible alpha-chains within fibrin, and (2) binding to an oriented substrate pair effectively lowers the entropic barrier to formation of a Michaelis complex and thus greatly enhances the rate of catalysis, e.g., for gamma-chain pairs within the fibrin fibrils.

Proc Natl Acad Sci U S A, 1997 Feb 4, 94(3), 837 - 42
Selective packaging of cargo molecules into endoplasmic reticulum-derived COPII vesicles; Campbell JL et al.; Coated vesicles transport proteins from the endoplasmic reticulum (ER) to the Golgi apparatus . The formation of transport vesicles in vitro requires the incubation of an ER-membrane fraction with three protein fractions collectively known as coat protein II (COPII; Sar1p, Sec23p/Sec24p, and Sec13p/Sec31p) . We used this assay to investigate how targeting {v-SNARE, vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor}, putative adapter (e.g., Emp24p), and cargo molecules are captured into ER-derived COPII vesicles . Analysis of fusion proteins strongly suggests that the cytoplasmic domain of the v-SNARE protein Sec22p is required for its packaging into ER-derived COPII vesicles . We examined the packaging requirements for various molecules by individually titrating each of the COPII components . More Sar1p (the GTP-binding protein that initiates vesicles budding) is needed to package the membrane-associated v-SNAREs and Emp24p than is needed to package the soluble secretory protein glycosylated pro-alpha-factor (gp alphaF) . Microsomes prepared from a strain overproducing Sec12p (the nucleotide exchange protein that recruits Sar1p to the ER) produce vesicles containing gp alphaF without the addition of exogenous Sar1p, whereas the v-SNAREs and Emp24p are not efficiently packaged under these conditions . Addition of Sar1p to these microsomes leads to increased packaging of v-SNAREs and Emp24p with no increase in the packaging of gp alphaF . Finally, we show that membranes prepared from strains with mutations in the SEC16 gene are more defective for the packaging of v-SNARE molecules and Emp24p than they are for the packaging of gp alphaF . These results point to the possibility that diverse signals or adapters participate in the capture of secretory and membrane cargo molecules into COPII transport vesicles.

Proc Natl Acad Sci U S A, 1997 Feb 4, 94(3), 820 - 5
The Dr1/DRAP1 heterodimer is a global repressor of transcription in vivo; Kim S et al.; A general repressor extensively studied in vitro is the human Dr1/DRAP1 heterodimeric complex . To elucidate the function of Dr1 and DRAP1 in vivo, the yeast Saccharomyces cerevisiae Dr1/DRAP1 repressor complex was identified . The repressor complex is encoded by two essential genes, designated YDR1 and BUR6 . The inviability associated with deletion of the yeast genes can be overcome by expressing the human genes . However, the human corepressor DRAP1 functions in yeast only when human Dr1 is coexpressed . The yDr1/Bur6 complex represses transcription in vitro in a reconstituted RNA polymerase II transcription system . Repression of transcription could be overcome by increasing the concentration of TATA-element binding protein (TBP) . Consistent with the in vitro results, overexpression of YDR1 in vivo resulted in decreased mRNA accumulation . Furthermore, YDR1 overexpression impaired cell growth, an effect that could be rescued by overexpression of TBP . In agreement with our previous studies in vitro, we found that overexpression of Dr1 in vivo also affected the accumulation of RNA polymerase III transcripts, but not of RNA polymerase I transcripts . Our results demonstrate that Dr1 functions as a repressor of transcription in vivo and, moreover, directly targets TBP, a global regulator of transcription.

Proc Natl Acad Sci U S A, 1997 Feb 4, 94(3), 790 - 5
Transcriptional repression at a distance through exclusion of activator binding in vivo; Shimizu M et al.; The yeast repressor Rme1p acts from distant binding sites to block transcription of the chromosomal IME1 gene . Rme1p can also repress the heterologous CYC1 promoter when Rme1p binding sites are placed 250-300 bp upstream of CYC1 transcriptional activator binding sites (UAS1 and UAS2) . Here, in vivo footprinting studies indicate that Rme1p acts over this distance by preventing the binding of the CYC1 transcriptional activators to UAS1 and UAS2 . Inhibition of activator binding by Rme1p has the same genetic requirements as repression: both depend upon sequences flanking the Rme1p binding sites and upon Rgr1p and Sin4p, two subunits of the RNA polymerase II-associated Mediator complex that are required for normal nucleosome density . Thus Rme1p may alter chromatin to prevent binding of transcriptional activators to distant DNA sequences.

Biochem Biophys Res Commun, 1997 Feb 3, 231(1), 227 - 30
In vitro kinetics of two human CYP1A1 variant enzymes suggested to be associated with interindividual differences in cancer susceptibility; Persson I et al.; A genetic polymorphism (A4889-->G) in the human CYP1A1 gene which creates an Ile462-->Val amino acid substitution has been suggested to cause altered enzymatic properties of CYP1A1 . Since several epidemiological studies have shown an association between the CYP1A1-Val allele and lung cancer, we considered it of importance to evaluate the in vitro kinetic properties of the two CYP1A1 variants after expression of each cDNA in yeast . No differences were found in K(m) or Vmax for CYP1A1 dependent O-dealkylation of ethoxyresorufin and 3-hydroxylation of benzo(a)pyrene between the two variants . The data indicate that the Ile/Val polymorphism in human CYP1A1 is not functionally important.

EMBO J, 1997 Feb 3, 16(3), 639 - 50
A role for DNA primase in coupling DNA replication to DNA damage response; Marini F et al.; The temperature-sensitive yeast DNA primase mutant pri1-M4 fails to execute an early step of DNA replication and exhibits a dominant, allele-specific sensitivity to DNA-damaging agents . pri1-M4 is defective in slowing down the rate of S phase progression and partially delaying the G1-S transition in response to DNA damage . Conversely, the G2 DNA damage response and the S-M checkpoint coupling completion of DNA replication to mitosis are unaffected . The signal transduction pathway leading to Rad53p phosphorylation induced by DNA damage is proficient in pri1-M4, and cell cycle delay caused by Rad53p overexpression is counteracted by the pri1-M4 mutation . Altogether, our results suggest that DNA primase plays an essential role in a subset of the Rad53p-dependent checkpoint pathways controlling cell cycle progression in response to DNA damage.

EMBO J, 1997 Feb 3, 16(3), 555 - 65
Histone acetyltransferase activity and interaction with ADA2 are critical for GCN5 function in vivo; Candau R et al.; Yeast GCN5 is one component of a putative adaptor complex that includes ADA2 and ADA3 and functionally connects DNA-bound transcriptional activators with general transcription factors . GCN5 possesses histone acetyltransferase (HAT) activity, conceptually linking transcriptional activation with enzymatic modification at chromatin . We have identified the minimal catalytic domain within GCN5 necessary to confer HAT activity and have shown that in vivo activity of GCN5 requires this domain . However, complementation of growth and transcriptional activation in gcn5- cells required not only the HAT domain of GCN5, but also interaction with ADA2 . The bromodomain in GCN5 was dispensable for HAT activity and for transcriptional activation by strong activators; however, it was required for full complementation in other assays . Fusion of GCN5 to the bacterial lexA DNA binding domain activated transcription in vivo, and required both the HAT domain and the ADA2 interaction domain . These results suggest that both functions of GCN5, HAT activity and interaction with ADA2, are necessary for targeting and acetylation of nucleosomal histones.






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