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 g