Yeasts

Gene, 1996 Oct 17, 176(1-2), 211 - 4
Isolation and characterization of a novel secretory protein, stromal cell-derived factor-2 (SDF-2) using the signal sequence trap method; Hamada T et al.; With use of the signal sequence trap method, we isolated a cDNA encoding a novel secretory protein, SDF-2, from the mouse stromal cell line, ST2 . The human homologue of SDF-2 was also isolated . The amino acid (aa) sequences deduced from both the clones were conserved more than 92% . The chromosomal localization of the human SDF-2 gene was mapped to 17q11.2 . The aa sequence of SDF-2 shows similarity to those of yeast dolichyl phosphate-D-mannose:protein mannosyltransferases, Pmt1p {Strahl-Bolsinger et al . (1993) Proc . Natl . Acad . Sci . USA 90, 8164-8168} and Pmt2p {Lussier et al . (1995) J . Biol . Chem . 270, 2770-2775}, whose activities have not been detected in higher eukaryotes.

Mol Gen Genet, 1996 Oct 16, 252(5), 503 - 9
Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis; Spellig T et al.; Pathogenic development of Ustilago maydis, the causative agent of corn smut disease, is a multistep process . Compatible yeast-like cells fuse and this generates the infectious dikaryon which grows filamentously . Having entered the plant the dikaryon induces tumors in its host in which massive proliferation of fungal material, karyogamy and spore formation occur . In order to follow fungal development from the initial steps to the final stage we have expressed the green fluorescent protein (GFP) from Aequorea victoria as a vital marker in U . maydis and demonstrate that GFP-tagged strains can be used to study host-pathogen interactions in vivo.

Genomics, 1996 Oct 15, 37(2), 200 - 10
Analysis of three deletion breakpoints in Xp21.1 and the further localization of RP3; Brown J et al.; The gene responsible for X-linked retinitis pigmentosa (xlRP) in Xp21.1 (RP3) was initially localized by deletion analysis to within a 150- to 170-kb region between the CYBB locus and the proximal deletion junction (BBJPROX) from a patient, BB, who suffered from Duchenne muscular dystrophy (DMD), McLeod syndrome, chronic granulomatous disease (CGD), and xlRP . This gene has recently been isolated and was found to be located outside and 400 kb proximal to the BB deletion . Further analysis of BBJPROX has identified the breakpoint junction sequence, showing that it occurs within an Alu repetitive element on the proximal side but with no significant homology to the distal sequence in dystrophin intron 30 . Analysis of an overlapping deletion in patient NF, who suffered from DMD, CGD, and McLeod syndrome, shows that this deletion is within 4 kb but extends centromeric to BBJPROX, consistent with the location of RP3 outside the BB deletion region . A sequence with strong homology to a THE-1 transposon-like element was identified 7-13 kb from the proximal BB and NF breakpoints . These elements have been implicated in several highly unstable genomic regions . A third overlapping deletion, in a patient, SB, who suffered from CGD, McLeod syndrome, and xlRP, has here been shown to extend 380 kb proximal to the NF breakpoint, consistent with the finding that RP3 lies outside the BB deletion . This deletion has now been shown to disrupt the RP3 (RPGR) gene . The reason for the retinitis pigmentosa phenotype in patient BB remains unclear, but the most likely explanations include a long-range chromosomal position effect, a small secondary rearrangement, and the presence of a coincident autosomal form of retinitis pigmentosa.

Nucleic Acids Res, 1996 Oct 15, 24(20), 4034 - 41
Ordered shotgun sequencing of a 135 kb Xq25 YAC containing ANT2 and four possible genes, including three confirmed by EST matches; Chen CN et al.; Ordered shotgun sequencing (OSS) has been successfully carried out with an Xq25 YAC substrate . yWXD703 DNA was subcloned into lambda phage and sequences of insert ends of the lambda subclones were used to generate a map to select a minimum tiling path of clones to be completely sequenced . The sequence of 135 038 nt contains the entire ANT2 cDNA as well as four other candidates suggested by computer-assisted analyses . One of the putative genes is homologous to a gene implicated in Graves' disease and it, ANT2 and two others are confirmed by EST matches . The results suggest that OSS can be applied to YACs in accord with earlier simulations and further indicate that the sequence of the YAC accurately reflects the sequence of uncloned human DNA.

Eur J Biochem, 1996 Oct 15, 241(2), 564 - 71
Cloning and characterization of cDNA for adenosine kinase from mammalian (Chinese hamster, mouse, human and rat) species . High frequency mutants of Chinese hamster ovary cells involve structural alterations in the gene; Singh B et al.; The enzyme adenosine kinase constitutes the major purine nucleoside phosphorylating activity in mammalian cells . In view of its central role in adenosine metabolism, which is an important physiological regulator, an understanding of the primary structure of adenosine kinase is of much interest . Using microsequence information from peptides derived from purified Syrian hamster liver enzyme, we have succeeded in isolating full length cDNA clones encoding adenosine kinase from Chinese hamster ovary cells and mouse 3T3 cells . The open reading frames in these clones consist of 334 and 335 amino acids and encode proteins of molecular masses 37364 Da and 37489 Da, respectively . In addition, the coding and upstream sequences for adenosine kinase from human (HeLa cells) and rat liver have also been cloned and sequenced . Transfection of an adenosine-kinase-deficient mutant (selected for resistance to the adenosine analog toyocamycin) of Chinese hamster ovary cells with a plasmid containing the cloned adenosine kinase cDNA, leads to regaining of adenosine kinase activity in the transformed cell . The adenosine kinase transformants also simultaneously lost their toyocamycin resistance and became similarly sensitive to the analog as the parental wild-type Chinese hamster ovary cells . The cloned adenosine kinase cDNA was also used to examine structural changes in mutants affected in adenosine kinase . In Chinese hamster ovary cells, one type of mutant that lacks adenosine kinase activity and displays high degree of resistance to various adenosine analogs, is obtained at an unusually high spontaneous frequency (10(-4)-10(-3)) . Results of Southern and northern-blot analysis provide evidence that this group of mutants involves gross structural alterations affecting the adenosine kinase gene . Such structural alterations are not observed in another type of mutant which exhibits increased resistance only to C-adenosine analogs . Sequence similarity searches indicate that several of the bacterial and yeast sugar kinases (ribokinase, fructokinase and inosine-guanosine kinase) exhibit limited but significant similarity to the mammalian adenosine kinase . The sequence similarity data support the possibility that adenosine kinase shares a common evolutionary ancestor with these protein sequences.

Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11837 - 41
Molecular cytogenetic delineation of a novel critical genomic region in chromosome bands 11q22.3-923.1 in lymphoproliferative disorders; Stilgenbauer S et al.; Aberrations of the long arm of chromosome 11 are among the most common chromosome abnormalities in lymphoproliferative disorders (LPD) . Translocations involving BCL1 at 11q13 are strongly associated with mantle cell lymphoma . other nonrandom aberrations, especially deletions and, less frequently, translocations, involving bands 11q21-923 have been identified by chromosome banding analysis . To date, the critical genomic segment and candidate genes involved in these deletions have not been identified . In the present study, we have analyzed tumors from 43 patients with LPD (B-cell chronic lymphocytic leukemia, n = 40; mantle cell lymphoma, n = 3) showing aberrations of bands 11q21-923 by fluorescence in situ hybridization . As probes we used Alu-PCR products from 17 yeast artificial chromosome clones spanning chromosome bands 11q14.3-923.3, including a panel of yeast artificial chromosome clones recognizing a contiguous genomic DNA fragment of approximately 9-10 Mb in bands 11q22.3-923.3 . In the 41 tumors exhibiting deletions, we identified a commonly deleted segment in band 11q22.3-923.1; this region is approximately 2-3 Mb in size and contains the genes coding for ATM (ataxia telangiectasia mutated), RDX (radixin), and FDX1 (ferredoxin 1) . Furthermore, two translocation break-points were localized to a 1.8-Mb genomic fragment contained within the commonly deleted segment . Thus, we have identified a single critical region of 2-3 Mb in size in which 11q14-923 aberrations in LPD cluster . This provides the basis for the identification of the gene(s) at 11q22.3-923.1 that are involved in the pathogenesis of LPD.

Blood, 1996 Oct 15, 88(8), 3109 - 15
Minimal region of loss at 13q14 in B-cell chronic lymphocytic leukemia; Bullrich F et al.; Allelic loss at nonrandom chromosomal sites is thought to mark the position of tumor suppressor genes involved in the pathogenesis and progression of human malignancies . Solid tumors in particular have been found to harbor multiple genetic changes resulting in loss of function mutations . Tumor suppressor genes have also been found to be involved in the progression of lymphoid tumors . Previous reports have suggested the involvement of a tumor suppressor gene located on the long arm of chromosome 13, between the retinoblastoma (RB) and D13S25 loci, in the pathogenesis and or progression of more than 40% of B-cell chronic lymphocytic leukemia (B-CLL), a common lymphoid malignancy whose molecular etiology remains largely unknown . In the present study, we report the construction and characterization of a YAC contig spanning a region of approximately 3 cM between the RB gene and the D13S31 locus . We also screened 60 paired normal/tumor B-CLL samples for allelic loss on chromosome 13 with nine microsatellite markers located between RB and D13S25 . This analysis has allowed us to narrow the smallest region of loss to a segment of 550 kb located between the 206XF12 and D13S25 markers.

FEMS Microbiol Lett, 1996 Oct 15, 144(1), 67 - 71
Utilization of acetonitrile and other aliphatic nitriles by a Candida famata strain; Linardi VR et al.; A variant of a yeast strain identified as Candida famata isolated from gold mine effluent was able to grow on acetonitrile, acrylonitrile, butyronitrile, isobutyronitrile, methacrylnitrile, propionitrile, succinonitrile, valeronitrile, acetamide, isobutyamide, and succinamide as sole nitrogen source, after acclimatization . The yeast grew on acetonitrile and acetamide at concentrations up to 4% . The utilisation of acetonitrile and acetamide by the C . famata strain probably involves hydrolysis in a two-step reaction mediated by both inducible and intracellular nitrile hydratase and amidase.

Biochem Biophys Res Commun, 1996 Oct 14, 227(2), 311 - 7
Disulfide bonds are necessary for structure and activity in Aspergillus ficuum phytase; Ullah AH et al.; The function of disulfide bonds in Aspergillus ficuum phytase was elucidated by unfolding studies, using guanidinium hydrochloride (Gu.HCl) as denaturant . Although the enzyme is totally inactivated by 0.8 M Gu.HCl, at pH 5.0, the active conformation is instantaneously restored by 0.6 M Gu.HCl, at pH 5.0 . Conditions which would permit refolding of phytase are completely negated by 10 mM beta-mercaptoethanol and causes its catalytic demise at pH 7.5 . Assay of free thiols using Ellman's reagent indicates that none of the thiols in the ten cysteines in phytase are free; five disulfide bonds were predicted for the enzyme . Sequence comparison of mold phytases and yeast acid phosphatases indicates four conserved cysteines . Thus, disulfide bonds play an important role in the folding of fungal phytase; any perturbation of the process of its formation causes an altered three-dimensional structure that is inconsistent with catalytic activity.

Science, 1996 Oct 11, 274(5285), 242 - 6
Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores; Chen RH et al.; The spindle assembly checkpoint delays anaphase until all chromosomes are attached to a mitotic spindle . The mad (mitotic arrest-deficient) and bub (budding uninhibited by benzimidazole) mutants of budding yeast lack this checkpoint and fail to arrest the cell cycle when microtubules are depolymerized . A frog homolog of MAD2 (XMAD2) was isolated and found to play an essential role in the spindle assembly checkpoint in frog egg extracts . XMAD2 protein associated with unattached kinetochores in prometaphase and in nocodazole-treated cells and disappeared from kinetochores at metaphase in untreated cells, suggesting that XMAD2 plays a role in the activation of the checkpoint by unattached kinetochores . This study furthers understanding of the mechanism of cell cycle checkpoints in metazoa and provides a marker for studying the role of the spindle assembly checkpoint in the genetic instability of tumors.

Gene, 1996 Oct 10, 175(1-2), 233 - 40
Molecular cloning of polybromo, a nuclear protein containing multiple domains including five bromodomains, a truncated HMG-box, and two repeats of a novel domain; Nicolas RH et al.; A number of transcription factors that act as adaptor proteins have been found to contain an 87 amino acid domain called the bromodomain . In a study to identify and characterise bromodomain proteins expressed in chicken cells, a novel gene has been isolated which encodes five repeats of the bromodomain . In addition, the encoded protein, termed polybromo, contains four other domains: an unusual truncated HMG box, two repeats of a novel domain which we term the BAH domain and a sequence related to a region within the regulatory domain of the DNA cytosine-5 methyltransferase enzyme . Polybromo was found to be related to a yeast protein U19102 which has two bromo domains, a BAH domain and the DNA methyltransferase-related sequence . Antibodies that were raised against polybromo were used in confocal microscopy analysis to show that the 180-kDa polybromo protein is located within the nucleus but excluded from the nucleolus . Gel filtration analysis of nuclear extracts demonstrate that polybromo is part of a large complex with a mass of approximately 2 million dalton.

Biochemistry, 1996 Oct 8, 35(40), 13157 - 64
ATP-binding site of human brain hexokinase as studied by molecular modeling and site-directed mutagenesis; Zeng C et al.; The interaction of ATP with the active site of hexokinase is unknown since the crystal structure of the hexokinase-ATP complex is unavailable . It was found that the ATP binding site of brain hexokinase is homologous to that of actin, heat shock protein hsc70, and glycerol kinase . On the basis of these similarities, the ATP molecule was positioned in the catalytic domain of human brain hexokinase, which was modeled from the X-ray structure of yeast hexokinase . Site-directed mutagenesis was performed to test the function of residues presumably involved in interaction with the tripolyphosphoryl moiety of ATP . Asp532, which is though to be involved in binding the Mg2+ ion of the MgATP2- complex, was mutated to Lys and Glu . The kcat values decreased 1000- and 200-fold, respectively, for the two mutants . Another residue, Thr680 was proposed to interact with the gamma-phosphoryl group of ATP through hydrogen bonds and was mutated to Val and Ser . The kcat value of the Thr680Val mutant decreased 2000-fold, whereas the kcat value of the Thr680Ser decreased only 2.5-fold, implying the importance of the hydroxyl group . The Km and dissociation constant values for either ATP or glucose of all the above mutants showed little or no change relative to the wild-type enzyme . The Ki values for the glucose 6-phosphate analogue 1,5-anhydroglucitol 6-phosphate, were the same as that of the wild-type enzyme, and the inhibition was reversed by inorganic phosphate (Pi) for all four mutants . The circular dichroism spectra of the mutants were the same as that of the wild-type enzyme . The results from the site-directed mutagenesis demonstrate that the presumed interactions of investigated residues with ATP are important for the stabilization of the transition state.

J Mol Biol, 1996 Oct 4, 262(4), 559 - 74
Protein globularization during folding . A study by synchrotron small-angle X-ray scattering; Semisotnov GV et al.; Various conformational states of polypeptide chains were investigated by synchrotron small-angle X-ray scattering (SAXS) . SAXS patterns of proteins and model polypeptides in globular states (native and "molten globule") and in non-globular states (unfolded protein as well as randomly coiled, partially alpha-helical and partially beta-structural synthetic polypeptides) were analyzed in terms of Guinier and Kratky plots . Large differences in the SAXS pattern have been found between globular and non-globular conformations of the polypeptide chains, and they have been interpreted in terms of differences in the shape and size of the globular and non-globular scatterers with the same molecular mass . The equilibrium and time-resolved unfolding curves of bovine carbonic anhydrase and yeast phosphoglycerate kinase were monitored by integrated SAXS intensity, and were found to be coincident with the curves measured by other physicochemical techniques, such as tryptophan fluorescence and peptide circular dichroism spectra . The intermolecular association of the protein "molten globule"-like intermediates accumulated during the guanidine hydrochloride-induced unfolding of bovine carbonic anhydrase has been investigated by various SAXS parameters . It has been shown that the integrated SAXS intensity is much less sensitive to the protein intermolecular association than the zero angle intensity and the radius of gyration . We propose the integrated SAXS intensity as a global parameter which is particularly appropriate for fast kinetic studies of protein coil to globule transitions . Time-resolved refolding curves of the above proteins were monitored by the integrated SAXS intensity to investigate the globularization process in protein folding . Two fast kinetic processes for bovine carbonic anhydrase and two fast (each within two seconds) as well as two slow (within 500 seconds) kinetic processes for yeast phosphoglycerate kinase have been recorded . The kinetic processes reflect both protein intramolecular globularization and its intermolecular association.

Cell, 1996 Oct 4, 87(1), 65 - 73
Requirement for PCNA in DNA mismatch repair at a step preceding DNA resynthesis; Umar A et al.; A two-hybrid system was used to screen yeast and human expression libraries for proteins that interact with mismatch repair proteins . PCNA was recovered from both libraries and shown in the case of yeast to interact with both MLH1 and MSH2 . A yeast strain containing a mutation in the PCNA gene had a strongly elevated mutation rate in a dinucleotide repeat, and the rate was not further elevated in a strain also containing a mutation in MLH1 . Mismatch repair activity was examined in human cell extracts using an assay that does not require DNA repair synthesis . Activity was inhibited by p21WAF1 or a p21 peptide, both of which bind to PCNA, and activity was restored to inhibited reactions by addition of PCNA . The data suggest a PCNA requirement in mismatch repair at a step preceding DNA resynthesis . The ability of PCNA to bind to MLH1 and MSH2 may reflect linkage between mismatch repair and replication and may be relevant to the roles of mismatch repair proteins in other DNA transactions.

J Biol Chem, 1996 Oct 4, 271(40), 24811 - 6
Molecular cloning of the cDNA and chromosome localization of the gene for human ubiquitin-conjugating enzyme 9; Wang ZY et al.; We report a novel human gene whose product specifically associates with the negative regulatory domain of the Wilms' tumor gene product (WT1) in a yeast two-hybrid screen and with WT1 in immunoprecipitation and glutathione S-transferase (GST) capture assays . The gene encodes a 17-kDa protein that has 56% amino acid sequence identity with yeast ubiquitin-conjugating enzyme (yUBC) 9, a protein required for cell cycle progression in yeast, and significant identity with other subfamilies of ubiquitin-conjugating enzymes . The human gene fully complements yeast that have a temperature-sensitive yUBC9 gene mutation to fully restore normal growth, indicating that we have cloned a functionally conserved human (h) homolog of yUBC9 . Transcripts of hUBC9 of 4.4 kilobases (kb), 2.8 kb, and 1.3 kb were found in all human tissues tested . A single copy of the hUBC9 gene was found and localized to human chromosome 16p13.3 . We conclude that hUBC9 retains striking structural and functional conservation with yUBC9 and suggest a possible link of the ubiquitin/proteosome proteolytic pathway and the WT1 transcriptional repressor system.

J Biol Chem, 1996 Oct 4, 271(40), 24675 - 83
Distinct domains of myocyte enhancer binding factor-2A determining nuclear localization and cell type-specific transcriptional activity; Yu YT; The myocyte enhancer binding factor 2 (MEF2) family of transcription factors plays an important role in the regulation of gene expression in multiple muscle cell types . Four mef2 genes (A, B, C, and D) have been identified in vertebrates . They share the conserved N-terminal MCM1-agamous-deficiens-serum response factor and MEF2 domains that determine DNA binding and dimerization functions . In this study, we have identified human MEF2A domains that control its nuclear localization and transcriptional activation function . Studies of subcellular localization of various truncated MEF2A proteins expressed in HeLa cells demonstrated that MEF2A contains a nuclear localization signal located at its C terminus within the peptide encompassing amino acids (aa) 472-507 . Examination of MEF2A mutants with sequential C-terminal deletions indicates that the region encompassing aa 321-472 is essential for transcriptional activity . Analysis of the transcriptional activity of fusion proteins consisting of different domains of MEF2A fused to the DNA binding domain of yeast transcription factor GAL4 revealed a major transcriptional activation domain located between aa 274 and 373 . Further studies demonstrated that this domain contains positive and negative regulatory subdomains that cooperate with one another to regulate the transcriptional activity of MEF2A in a cell-type discriminatory manner.

J Biol Chem, 1996 Oct 4, 271(40), 24583 - 9
c-Jun can mediate androgen receptor-induced transactivation; Bubulya A et al.; The proto-oncoprotein c-Jun forms as a heterodimer with c-Fos, the transcription factor AP-1 . AP-1 regulates transcription through transactivation, a process requiring DNA binding . Here we report an indirect mechanism by which c-Jun can regulate transcription via the androgen receptor . In this process, c-Jun is able to support androgen receptor-mediated transactivation in the absence of an interaction with c-Fos or any apparent DNA binding . This positive effect of c-Jun was dose-dependent . Both exogenously added and endogenously induced c-Jun are able to act on the androgen receptor . Transactivation by the androgen receptor can undergo self-squelching, and this was relieved by transfected c-Jun . Using a time-course experiment, we provide evidence that the c-Jun effect is primary . c-Fos is able to block human androgen receptor activity in both the absence and presence of transfected c-Jun . Using a modified form of the yeast two-hybrid system, we show in Cos cells that c-Jun can interact with the DNA binding domain/hinge region (CD regions) of the androgen receptor . Therefore, we propose that c-Jun functions as a mediator for androgen receptor-induced transactivation.

J Biol Chem, 1996 Oct 4, 271(40), 24564 - 8
Role of hydrophobic amino acids at beta85 and beta88 in stabilizing F helix conformation of hemoglobin S; Reddy LR et al.; Three Hb S variants containing Glu substitutions at Phe-beta85 and/or Leu-beta88 were expressed in yeast in an effort to evaluate the role of hydrophobic amino acids at these sites in stabilizing F helix conformation of Hb S . Helix stability of tetrameric Hb S betaF85E,betaL88E was measured by CD and compared with those of Hb S betaF85E, Hb S betaL88E, Hb A, and Hb S . The CD spectra of these Hb S variants were similar to those of Hb S and Hb A at 10 degrees C . However, changes in ellipticity at 222 nm for Hb S betaF85E in the CO form at 60 degrees C were about 15-fold greater than that of Hb S, while those for Hb S betaL88E and Hb S betaF85E,betaL88E were similar and about 30-fold greater than Hb S . Thermal stability measured by continuous scanning of spectral changes revealed the three Hb S variants were much more unstable than Hb S, and stability of Hb S betaF85E,betaL88E was similar to that of Hb S betaL88E rather than Hb S betaF85E . These results suggest that Glu insertion at both beta85 and beta88 makes heme insertion into the heme pocket more difficult; however, once inserted, stability of Hb S betaF85E, betaL88E is similar to Hb S betaL88E rather than Hb S betaF85E . Furthermore, these results suggest that both Phe-beta85 and Leu-beta88 are critical for F helix stabilization and that Glu insertion at beta88 leads to more destabilization than insertion at beta85.

J Biol Chem, 1996 Oct 4, 271(40), 24557 - 63
Polymerization of three hemoglobin A2 variants containing Val6 and inhibition of hemoglobin S polymerization by hemoglobin A2; Adachi K et al.; To understand determinants for hemoglobin (Hb) stability and Hb A2 inhibition of Hb S polymerization, three Valdelta6 Hb A2 variants (Hb A2 deltaE6V, Hb A2 deltaE6V,deltaQ87T, and Hb A2 deltaE6V, deltaA22E,deltaQ87T) were expressed in yeast, and stability to mechanical agitation and polymerization properties were assessed . Oxy forms of Hb A2 deltaE6V and Hb A2 deltaE6V,deltaQ87T were 2- and 1.6-fold, respectively, less stable than oxy-Hb S, while the stability of Hb A2 deltaE6V,deltaA22E,deltaQ87T was similar to that of Hb S, suggesting that Aladelta22 and Glndelta87 contribute to the surface hydrophobicity of Hb A2 . Deoxy Hb A2 deltaE6V polymerized without a delay time, like deoxy Hb F gammaE6V, while deoxy Hb A2 deltaE6V,deltaQ87T and deoxy Hb A2 deltaE6V,deltaA22E,deltaQ87T polymerized after a delay time, like deoxy Hb S, suggesting that beta87 Thr is required for the formation of nuclei . Deoxy Hb F gammaE6V,gammaQ87T showed no delay time and required a 3.5-fold higher concentration than deoxy Hb S for polymerization, suggesting that Thr effects on Valdelta6 Hb A2 and Valgamma6 Hb F variants are different . Mixtures of deoxy Hb S/Hb A2 deltaE6V,deltaQ87T polymerized, like deoxy Hb S, while polymerization of Hb S/Hb A2 deltaE6V mixtures was inhibited, like Hb S/Hb F gammaE6V mixtures . These results suggest alpha2betaSdelta6 Val, 87 Thr hybrids and Hb A2 deltaE6V,deltaQ87T participate in Hb S nucleation, while only 50% of alpha2betaSdelta6 Val hybrids and none of the Hb A2 deltaE6V participate . These findings are in contrast to those of mixtures of Hb S with Hb F gammaE6V or Hb F gammaE6V,Q87T, which both inhibit Hb S polymerization . Our results also suggest participation in nucleation of some alpha2betaSdelta hybrids in A2S mixtures but not alpha2betaSgamma hybrids in FS mixtures.

Biochem Biophys Res Commun, 1996 Oct 3, 227(1), 152 - 9
Evidence that the human homologue of a rat initiation factor-2 associated protein (p67) is a methionine aminopeptidase; Li X et al.; Previously, we cloned a human cDNA encoding a protein which has a 92% amino acid sequence identity to a rat initiation factor-2 associated protein (p67) . Rat p67 plays an important role in translational regulation by preventing the phosphorylation of the alpha subunit of initiation factor-2 . Interestingly, several lines of indirect evidence suggested that this protein may also function as a methionine aminopeptidase (MetAP) . To test this hypothesis, we expressed the human cDNA in a baculovirus system, purified it to homogeneity and characterized it . Using 13 different peptide substrates, we found that the human p67 has a similar substrate specificity with other MetAPs . Kinetic analyses revealed that the Kcat/K(m) values of the human MetAP on two representative substrates are similar to those of yeast and porcine MetAPs . Furthermore, we found that this enzyme, like other MetAPs, is also a cobalt-dependent metalloenzyme.

Biochem Biophys Res Commun, 1996 Oct 3, 227(1), 82 - 7
The metastasis suppressor candidate nucleotide diphosphate kinase NM23 specifically interacts with members of the ROR/RZR nuclear orphan receptor subfamily; Paravicini G et al.; We have cloned proteins that interact with the nuclear orphan receptor RZR beta using the yeast two-hybrid system . We identified, amongst a number of other genes, the nucleoside diphosphate kinase (NDPK)-2 also known as Nm23-2, c-myc regulatory factor PuF and differentiation inhibitory factor, RZR beta specifically interacts with Nm23-2 but not with the closely related tumor metastasis suppressor candidate gene product Nm23-1 . In contrast ROR alpha interacts with both Nm23 proteins . These findings were corroborated by in vitro interaction assays based on GST-pulldown experiments . With-n-myc we propose a candidate gene regulated by ROR alpha/RZR beta and Nm23, based on the finding that the respective DNA binding sites in the first intron are conserved in several mammalian species.

Nature, 1996 Oct 3, 383(6599), 453 - 7
A new group of conserved coactivators that increase the specificity of AP-1 transcription factors; Claret FX et al.; The Jun proteins are nuclear proteins that combine with Fos proteins to form a gene-regulatory protein, AP-1 . They have highly conserved DNA-binding and dimerization domains, resulting in almost identical sequence-recognition properties . Nevertheless, there are many indications that each Jun protein activates a distinct and only partially overlapping set of AP-1 target genes . Using the more variable activation domain of c-Jun as a bait, we identified a protein, JAB1, that interacts with c-Jun and JunD, but not with JunB or v-Jun . As a result, JAB1 selectively potentiates transactivation by only c-Jun or JunD . In vitro, JAB1 specifically stabilizes complexes of c-Jun or JunD with AP-1 sites and does not affect binding of either JunB or v-Jun . The amino-terminal half of JAB1 is very similar to the amino terminal region of Pad1 from fission yeast, which was identified genetically as a coactivator of a subset of AP-1 target genes . JAB1 and Pad1 are also functionally interchangeable . They define a new group of coactivators that increase the specificity of target gene activation by AP-1 proteins.

Biochim Biophys Acta, 1996 Oct 2, 1284(1), 9 - 12
Cloning and sequencing of the bovine cDNA encoding the mitochondrial tricarboxylate carrier protein; Iacobazzi V et al.; The tricarboxylate or citrate transporter protein (CTP) catalyzes the transport of citrate across the inner mitochondrial membrane by an exchange for malate or some other anionic metabolite . Using primers based on the rat liver cDNA sequence, overlapping cDNA clones encoding the bovine CTP were isolated from bovine liver poly(A+) cDNA . The entire bovine cDNA is 1151 bp in length with 5' and 3' untranslated regions of 7 and 204 bp, respectively . The open reading frame encodes the mature protein consisting of 298 amino acids, preceded by a presequence of 13 amino acids . The amino acid sequence of the mature bovine CTP is 95.6, 94.9, 32.2% identical to that of the citrate carrier from man, rat and yeast, respectively.

Cell Struct Funct, 1996 Oct, 21(5), 421 - 4
A role of cofilin/destrin in reorganization of actin cytoskeleton in response to stresses and cell stimuli; Yahara I et al.; 1 . Cofilin is an essential actin-regulating protein widely distributed in all eucaryotes . The structure and function of cofilin are conserved during evolution . 2 . Cofilin depolymerizes F-actin in vitro at alkaline pH and severs F-actin in vitro at pH lower than 7.3 . Overexpression of cofilin in viable cells induced bundles of actin filaments suggesting that the severing activity rather than the actin-depolymerizing or monomeric actin-sequestering activity is physiologically significant in vivo . 3 . The actin bundle formation induced by overexpression of cofilin is accompanied with an increase in cell motility of Dictyostelium cells . 4 . In higher vertebrates, the actin-binding activity of cofilin is negatively regulated by phosphorylation on its Ser-3 residue . The actin-binding activity is essential for yeast cells to grow . 5 . Stresses and various cell stimuli activate cofilin by inducing dephosphorylation of cofilin in resting vertebrate cells . 6 . Cofilin has an nuclear localization signal sequence and translocates into the nucleus together with actin in response to various stresses . Functional roles of cofilin/actin in the nucleus remain to be elucidated . 7 . Tertiary structure of destrin (cofilin) resembles that of gelsolin segment 1 and well explains its functions such as Ca(2+)-independent actin binding activity.

Semin Cancer Biol, 1996 Oct, 7(5), 299 - 306
Complementation tagging of cooperating oncogenes in knockout mice; Allen JD et al.; Gene disruption and overexpression in mice can be combined with proviral tagging to investigate the biochemical pathways in which oncogenes act . The phenomenon of oncogene collaboration permits a focussed genetic approach analogous to the strategies employed in suppressor screens in invertebrates and yeast . We illustrate here its application to investigating the action of the pim kinases in tumorigenesis . However, it should be possible to utilize this strategy for dissecting a much wider range of biochemical pathways in mammalian systems.

Bioorg Khim, 1996 Oct-Nov, 22(10-11), 870 - 4
{Precipitation of cytochromes c with complex cadmium iodide}; Zhuravleva DV et al.; Using cytochrome c from horse heart and the yeast candida valida as examples, it was shown that a complex anion, cadmium tetraiodide (CdI42-), precipitated proteins from aqueous solutions at the reagent concentrations below 50 mM . The composition and pH value of the solution, as well as the starting protein concentration, considerably influenced the precipitation . The results suggest that this reagent acts on the protein by a mechanism similar to the salting-out process . The ability to act at small concentrations is the advantage of CdI42- over conventional agents.

Med Parazitol (Mosk), 1996 Oct-Dec, (4), 43 - 5
{A dry nutrient medium for the cultivation of Leishmania promastigotes}; Osokina TI et al.; A dry medium formulation which provides the growth and reproduction of Leishmania promastigotes was designed . The medium has been prepared by using nondietary protein raw material, it contains domestic-production ingredients of that in high supply . The nutrient basis of the medium is enzymatic peptone (a source of amine nitrogen) . The growth factors of Leishmania (a yeast extract, vitamins, amino acids, a dry blood derivative), carbohydrates (an energy components of the medium), inorganic salts (a source of trace elements), which are selected in strictly defined quantitative ratios, meet the physiological requirements of Leishmania . The agent rules out the necessity of adding ex tempore blood, making the process for preparing the medium from the dry powder simple and easy-to-use.

Clin Genet, 1996 Oct, 50(4), 267 - 9
FISH characterization of the Xq21 breakpoint in a translocation carrier with premature ovarian failure; Riva P et al.; A panel of ordered YAC clones, isolated using STSs in the Xq13-Xq23 region, was used to characterize by Fluorescent In Situ Hybridization (FISH) the Xq21 breakpoint in a t(X;1)(q21;p34) translocation female with premature ovarian failure . The YAC 949E11 was found to span the breakpoint, but also to join the two non-overlapping YACs 36CB1 and 40AB3, proximal and distal, respectively, to the patient's Xq21 breakpoint.

Lett Appl Microbiol, 1996 Oct, 23(4), 253 - 6
Galacto-oligosaccharide production from lactose by Sirobasidium magnum CBS6803; Onishi N et al.; Galacto-oligosaccharide (Gal-OS) was produced from lactose by a yeast, Sirobasidium magnum CBS6803 . With toluene-treated resting cells, 136 mg ml-1 of Gal-OS was produced from 360 mg ml-1 of lactose at 50 degrees C for 42 h . Then, the yield of Gal-OS was increased by a culture method in which cell growth followed the enzymatic reaction: 224 mg ml-1 of Gal-OS was produced at 30 degrees C for 60 h . Finally, combination of the toluene-treated resting cells and glucose oxidase plus catalase was applied to improve productivity by the removal of a by-product, glucose, which inhibits the Gal-OS production, from the reaction mixture . In this case, 242 mg ml-1 of Gal-OS was produced at 50 degrees C for 42 h without cell growth . The structure of the major product was identified as 4'-galactosyl-lactose.

Plant Mol Biol, 1996 Oct, 32(1-2), 1 - 41
Splicing of precursors to mRNA in higher plants: mechanism, regulation and sub-nuclear organisation of the spliceosomal machinery; Simpson GG et al.; The removal of introns from pre-mRNA transcripts and the concomitant ligation of exons is known as pre-mRNA splicing . It is a fundamental aspect of constitutive eukaryotic gene expression and an important level at which gene expression is regulated . The process is governed by multiple cis-acting elements of limited sequence content and particular spatial constraints, and is executed by a dynamic ribonucleoprotein complex termed the spliceosome . The mechanism and regulation of pre-mRNA splicing, and the sub-nuclear organisation of the spliceosomal machinery in higher plants is reviewed here . Heterologous introns are often not processed in higher plants indicating that, although highly conserved, the process of pre-mRNA splicing in plants exhibits significant differences that distinguish it from splicing in yeast and mammals . A fundamental distinguishing feature is the presence of and requirement for AU or U-rich intron sequence in higher-plant pre-mRNA splicing . In this review we document the properties of higher-plant introns and trans-acting spliceosomal components and discuss the means by which these elements combine to determine the accuracy and efficiency of pre-mRNA processing . We also detail examples of how introns can effect regulated gene expression by affecting the nature and abundance of mRNA in plants and list the effects of environmental stresses on splicing . Spliceosomal components exhibit a distinct pattern of organisation in higher-plant nuclei . Effective probes that reveal this pattern have only recently become available, but the domains in which spliceosomal components concentrate were identified in plant nuclei as enigmatic structures some sixty years ago . The organisation of spliceosomal components in plant nuclei is reviewed and these recent observations are unified with previous cytochemical and ultrastructural studies of plant ribonuleoprotein domains.

Genet Anal, 1996 Oct, 13(4), 99 - 103
Identification and mapping of a putative bombesin receptor gene on human chromosome 17q21.3+; Kelavkar U et al.; A mouse bombesin receptor cDNA was used as a probe to screen a human P1 genomic library . Clone HBR1 was isolated and used to localize a putative human bombesin receptor gene (HBRKS) on human chromosome 17q21.3 by fluorescent in situ hybridization (FISH) . HBRKS was identified and mapped by polymerase chain reaction (PCR) amplification from a Yeast artificial chromosome (YAC) contig spanning 17q21-q23 . In addition, a few candidate genes were found by exon-trapping from HBR1.

J Med Genet, 1996 Oct, 33(10), 842 - 7
Precise localisation of 3p25 breakpoints in four patients with the 3p-syndrome; Drumheller T et al.; In patients with the 3p-syndrome, hemizygous deletion of 3p25-pter is associated with profound growth failure, characteristic facial features, and mental retardation . We performed a molecular genetic analysis of 3p25 breakpoints in four patients with the 3p- syndrome, and a fifth patient with a more complex abnormality, 46,XY,der(3)t(3;?)(p25.3;?) . EBV transformed lymphoblasts from each of the patients were initially characterised using fluorescent in situ hybridisation (FISH) and polymorphic microsatellite analyses . The 3p-chromosome from each patient was isolated from the normal chromosome 3 in somatic cell hybrid lines and subsequently analysed with polymorphic and monomorphic PCR amplifiable markers from 3p25 . The analysis clearly shows that all five breakpoints are distinct . Furthermore, we have identified yeast artificial chromosomes that cross the 3p25 breakpoints of all four 3p-patients . Two of the patients were deleted for the von Hippel-Lindau (VHL) tumour suppressor gene, although neither has yet developed evidence of VHL disease . The patient with the most centromeric breakpoint, between D3S1585 and D3S1263, had the most severe clinical phenotype including an endocardial cushion defect that was not observed in any of the four patients who had more telomeric breakpoints . This study should provide useful insights into critical regions within 3p25 that are involved in normal human growth and development.

Environ Health Perspect, 1996 Oct, 104(10), 1084 - 9
Identification of environmental chemicals with estrogenic activity using a combination of in vitro assays; Klotz DM et al.; Environmental chemicals that function as estrogens have been suggested to be associated with an increase in disease and dysfunctions in animals and humans . To characterize chemicals that may act as estrogens in humans, we have compared three in vitro assays which measure aspects of human estrogen receptor (hER)-mediated estrogenicity . Chemicals were first tested for estrogen-associated transcriptional activity in the yeast estrogen screen (YES) . This was created by expressing hER and two estrogen response elements linked to the lacZ gene in yeast . Second, chemicals that were tested in YES were then assayed for direct interaction with hER in a competition binding assay . Third, chemicals were tested in the estrogen-responsive MCF-7 human breast cancer cell line transiently transfected with a plasmid containing two estrogen response elements linked to the luciferase gene . Together, these assays have identified two metabolites of DDT, o,p'-DDD and p,p'-DDD, that have estrogenic activity . Interestingly, previous studies had reported that the DDD metabolites were nonestrogenic in whole animal models . Alachlor, the most frequently used herbicide in the United States, cis-nonachlor, and trans-nonachlor displayed weak estrogenic activity in the combined assays . The antifungal agent benomyl had no estrogenic activity . We propose that a combination of in vitro assays can be used in conjunction with whole animal models for a more complete characterization of chemicals with estrogenic activity.

Mol Cell Biol, 1996 Oct, 16(10), 5865 - 75
Two new members of the murine Sim gene family are transcriptional repressors and show different expression patterns during mouse embryogenesis; Ema M et al.; From a cDNA library of mouse skeletal muscle, we have isolated mouse Sim1 (mSim1) cDNA encoding a polypeptide of 765 amino acids with striking amino acid identify in basic helix-loop-helix (89% identify) and PAS (89 % identify) domains to previously identified mSim2, although the carboxy-terminal third of the molecule did not show any similarity to mSim2 or Drosophila Sim (dSim) . Yeast two-hybrid analysis and coimmunoprecipitation experiments demonstrated that both of the mSim gene products interacted with Arnt even more efficiently than AhR, a natural partner of Arnt, suggesting a functional cooperativity with Arnt . In sharp contrast with dSim having transcriptional-enhancing activity in the carboxy-terminal region, the two mSims possessed a repressive activity toward Arnt in the heterodimer complex . This is the first example of bHLH-PAS proteins with transrepressor activity, although some genetic data suggest that dSim plays a repressive role in gene expression (Z . Chang, D . Price, S . Bockheim, M . J . Boedigheimer, R . Smith, and A . Laughon, Dev . Biol . 160:315-322, 1993; D . M . Mellerick and M . Nirenberg, Dev . Biol . 171:306-316, 1995) . Whole-mount in situ hybridization showed restricted and characteristic expression patterns of the two mSim mRNAs in various tissues and organs during embryogenesis, such as those for the somite, the nephrogenic cord, and the mesencephalon (for mSim1) and those for the diencephalon, branchial arches, and limbs (for mSim2) . From sequence similarity and chromosomal localization, it is concluded that mSim2 is an ortholog of hSim2, which is proposed to be a candidate gene responsible for Down's syndrome . The sites of mSim2 expression showed an overlap with the affected regions of the syndrome, further strengthening involvement of mSim2 in Down's syndrome.

Acta Paediatr, 1996 Oct, 85(10), 1143 - 5
Selenium supply and glutathione peroxidase activity in breastfed Polish infants; Trafikowska U et al.; Selenium (Se) concentration in human milk in Poland is below 10 ng ml-1 and the Se intake by breastfed infants is about 6 micrograms day-1 . Supplementation of lactating mothers with selenium-enriched yeast increases rapidly and significantly the Se concentration and glutathione peroxidase activity in maternal blood components . Se concentration in milk is also significantly elevated . After 1 month the mean Se intakes by breastfed infants were greater than the recommended dietary allowance of 10 micrograms day-1 for infants from birth to 6 months of age.

Anticancer Drug Des, 1996 Oct, 11(7), 493 - 508
Biochemical and chemical characterization of phenylglyoxal bis(guanylhydrazone), an aromatic analogue of mitoguazone; Elo H et al.; Since little has been known about the properties of aromatic analogues of the antineoplastic agent methylglyoxal bis(guanylhydrazone) (MGBG), an investigation was performed on phenylglyoxal bis(guanylhydrazone) (PhGBG) . PhGBG competitively inhibited yeast adenosylmethionine decarboxylase (AdoMetDC) with a Ki of 65 microM . As compared to MGBG (Ki 0.23 microM), PhGBG is a much weaker inhibitor, being even weaker than the unsubstituted congener glyoxal bis(guanylhydrazone) (GBG, Ki 18 microM) . PhGBG inhibited porcine kidney diamine oxidase (DAO) non-competitively, being a more potent inhibitor (Ki 0.12 microM) than GBG (Ki 0.17 microM) or MGBG (Ki 0.33 microM) . Thus, PhGBG has an unfavourably high ratio of Ki(AdoMetDC)/Ki(DAO) for potential use for selectively inhibiting polyamine biosynthesis . This does not exclude the possibility that PhGBG or other aromatic congeners might have therapeutic value since the corresponding ratio of the antileukaemic congeners GBG and MGBG is also high as compared to many aliphatic non-antileukaemic analogues . The pKa1 and pKa2 values of PhGBG dication were found to be 6.39 +/- 0.02 and 8.64 +/- 0.02 respectively, their difference being distinctly larger than in the case of GBG or its C-alkylated analogues . This may result from decreased stability of the dication form, caused by the resonance effect or possibly by the inductive effect of the phenyl group . The species distribution of PhGBG (proportion of free base 5.5%, predominant species the monocation) at 37 degrees C resembles that of GBG and MGBG but is clearly different from that of non-antileukaemic C-alkylated analogues . These similarities suggest that PhGBG and its derivatives may be worth antitumour screening . Depending on the conditions used in the crystallization, three different types of crystals of PhGBG sulphate were obtained . Crystallography indicated that, in two of the types, the crystal consisted exclusively of the anti-anti isomer, i.e . the same isomer as has been observed in the case of GBG and its C-alkylated congeners . One crystal type, however, consisted of a different geometrical isomer (anti-syn), suggesting that PhGBG may isomerize more easily than its aliphatic analogues . Previous concepts on the isomerism of GBG and C-alkylated bis(guanylhydrazones) thus cannot be generalized to aromatic congeners . A theory based on resonance, inductive and hyperconjugative effects and electron transfers is presented that is capable of explaining the formation of the two geometrical isomers of PhGBG that were experimentally observed . A similar theory, based on hyperconjugation of C-F bonds, is presented that is capable of explaining the previous finding of the formation of the anti-syn isomer of trifluoromethylglyoxal bis(guanylhydrazone) (CF3GBG) . Like that of CF3GBG, the anti-syn isomer of the PhGBG dication is stabilized by an internal hydrogen bond . The lack of structural rigidity may affect the biological properties of PhGBG, e.g . its ability to inhibit AdoMetDC.

Genomics, 1996 Oct 1, 37(1), 24 - 8
The B-lymphocyte maturation promoting transcription factor BLIMP1/PRDI-BF1 maps to D6S447 on human chromosome 6q21-q22.1 and the syntenic region of mouse chromosome 10; Mock BA et al.; The human PRDI-BF1 or BLIMP1 gene and its mouse homolog Blimp1 are members of the recently realized PR domain family that includes the retinoblastoma interacting zinc finger gene RIZ and the MDS1-EVI1 leukemia cancer gene . The specific high-level expression of Blimp1 in late B and plasma cells, its induction during B-cell differentiation, and its ability to drive B-cell maturation suggest that this gene may play a role in the differentiation and pathogenesis of B cells . We have now mapped the physical location of BLIMP1 near the marker D6S447 on human chromosome 6q21-q22.1; we have also mapped Blimp1 to mouse Chromosome 10 at 14 cM distal to the Myb locus and to a region homologous to the location of BLIMP1 . Deletions of the 6q21-q22 region are common in several human malignancies, particularly in B-cell non-Hodgkin lymphoma (B-NHL) . The data led us to suggest that BLIMP1 may be a candidate B-NHL tumor suppressor gene.

Genomics, 1996 Oct 1, 37(1), 9 - 18
High-density radiation hybrid map of human chromosome 18 and contig of 18p; Giacalone J et al.; A high-resolution radiation hybrid map of human chromosome 18 has been developed by testing DNA samples of 92 radiation hybrids (RH) from a previously characterized chromosome 18-specific RH panel . Half of the 159 STS markers were tested on RH DNA amplified by primer extension preamplification . This map includes 20 genes and 95 polymorphic markers, most of which have heterozygosity frequencies greater than 65% and, therefore, allow integration with genetic maps . The framework map consists of 137 markers ordered with odds of 1000:1 or better and spaced on average 580 kb apart . A YAC contig map of 18p was generated independently by STS content mapping of YACs using the same primers as for RH mapping . The RH map and the contig map are concordant.

Genes Chromosomes Cancer, 1996 Oct, 17(2), 108 - 17
Fluorescence in situ hybridization deletion mapping at 4p16.3 in bladder cancer cell lines refines the localisation of the critical interval to 30 kb; Bell SM et al.; An allelotype analysis of transitional cell carcinoma of the bladder identified loss of heterozygosity (LOH) on chromosome arm 4p in 22% of tumours . In a more detailed LOH study of 178 bladder carcinomas, a 750 kb common region of deletion was identified between the markers D4S43 and D4S127 just telomeric to the Huntington disease locus . To refine this region of deletion at 4p16.3, we have carried out detailed fluorescence in situ hybridisation (FISH) analysis of 12 bladder cancer cell lines by using a chromosome 4 centromeric probe combined with a series of cosmid probes from contigs spanning the 750 kb region of deletion . A common 30 kb region of deletion was identified at 4p16.3 in over one-third of the bladder cancer cell lines analysed . The present study has refined the localisation of the critical region of deletion from 750 kb to approximately 30 kb, providing a precise starting point for positional cloning of the gene(s) involved in bladder cancer from within a very gene-rich region on chromosome band 4p16.3 . This study demonstrates that FISH can be used for fine deletion mapping of potential tumour suppressor gene regions . The utilisation of FISH analysis to map chromosomal deletions should facilitate positional cloning of other genes as bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) contigs of the human genome are established.

Genes Chromosomes Cancer, 1996 Oct, 17(2), 78 - 87
Detection of chromosomal DNA gains and losses in testicular germ cell tumors by comparative genomic hybridization; Korn WM et al.; To extend the results of conventional cytogenetic analysis of testicular germ cell tumors (TGCTs), we applied the new molecular cytogenetic method of comparative genomic hybridization (CGH), which enables the detection of chromosomal imbalances without the need for dividing cells . DNA from II TGCTs was studied by CGH . In all tumors examined, gain of 12p, mostly of the whole p arm, could be demonstrated . However, in three tumors, an amplification of 12p material restricted to the chromosomal bands 12p11.2-p12.1 was found . Further fluorescence in situ hybridization (FISH) analysis using a yeast artificial chromosome (YAC) that was previously mapped to that region revealed multiple copies of that chromosomal segment in interphase nuclei of these tumors . This finding is an important clue to the localization of candidate protooncogenes at 12p involved in TGCTs . Gains of small chromosomal regions at 2p, 4q, 6p, and 19p were also detected recurrently . Furthermore, gains of chromosomes 8, 14, 21, and X as well as loss of chromosome 13 were frequent findings . In conclusion, CGH provides new insights into genetic alterations of TGCTs . By using CGH, chromosomal subregions could be identified that may harbor genes involved in the pathogenesis of this malignancy.

Immunol Cell Biol, 1996 Oct, 74(5), 457 - 64
Breast cancer immunotherapy: current status and future prospects; Apostolopoulos V et al.; The development of an immunotherapeutic approach to cancer is the concern for many immunologists, but despite the impressive progress over the past decade, such as the identification of tumour antigens and antigenic peptides as potential targets, there are still many obstacles in eliciting an effective immune response to eradicate cancer . Mucins have attracted interest as potential targets for immunotherapy in the development of vaccines for cancers expressing Mucin1 (MUC1; e.g . breast, pancreas, ovary etc.) . All of the identified targets for cancer, including MUC1, are normal proteins; however MUC1 expressed on tumours can be considered as tumour specific due to their overexpression, altered glycosylation and its ubiquitous distribution on the cell surface rather than at the secretory pole in adenocarcinomas . These observations have led to the development of several different approaches to immunize against breast cancer using synthetic carbohydrates or peptides conjugated to carriers and given together with a variety of adjuvants to elicit the appropriate immune response . Mannan, a polymannose carbohydrate isolated from the cell wall of yeast, is an appropriate and effective protein carrier for eliciting a cellular (T1-type) or humoral (T2-type) immune response depending on the mode of conjugation (oxidized or reduced) . In addition, mannan holds promise and opens many avenues as a carrier for vaccine development for other antigens . Several clinical trials are in progress to evaluate the immunogenicity of MUC1 and its suitability as to use for immunotherapy/vaccine for breast cancer.

Genome Res, 1996 Oct, 6(10), 1002 - 12
Utilization of FISH in positional cloning: an example on 13q22; Laan M et al.; In positional cloning the initial assignment of a gene to a specific chromosomal locus is followed by physical mapping of the critical region . The construction of a high-resolution physical map still involves considerable effort . However, new high-resolution fluorescence in situ hybridization (FISH) techniques have facilitated this process substantially . Here we summarize a strategy that combines a spectrum of FISH techniques {metaphase, interphase, mechanically stretched chromosomes (MSCs), and fiber-FISH on free chromatin} for the construction and characterization of a high-resolution physical map for a positional cloning project . The chromosomal region 13q22, containing the locus of the variant form of the neuronal ceroid lipofuscinosis (vLINCL, CLN5) disease, serves here as an example for this process . We used metaphase FISH to exclude positionally a candidate gene, to refine the locus to 13q22, and to analyze the possible chimerism of the YACs in the region . Both metaphase and interphase FISH techniques were applied to determine the low-resolution distances between the restricting markers . FISH using MSCs confirmed the centromeric-telomeric order of the clones and facilitated the estimation of the size of the gaps between the clones . Finally, fiber-FISH was found to be the method of choice for the construction of an accurate high-resolution map of the contig established over the restricted region . Thus, FISH techniques in combination with genetic mapping data enabled the refinement of the initial 4-cM region to a high-resolution map of only 400 kb in length . Here the FISH strategy replaced the need for many laborious traditional physical mapping methods, e.g., pulsed-field gel electrophoresis.

Genome Res, 1996 Oct, 6(10), 943 - 55
An integrated YAC map of the human X chromosome; Roest Crollius H et al.; The human X chromosome is associated with a large number of disease phenotypes, principally because of its unique mode of inheritance that tends to reveal all recessive disorders in males . With the longer term goal of identifying and characterizing most of these genes, we have adopted a chromosome-wide strategy to establish a YAC contig map . We have performed > 3250 inter Alu-PCR product hybridizations to identify overlaps between YAC clones . Positional information associated with many of these YAC clones has been derived from our Reference Library Database and a variety of other public sources . We have constructed a YAC contig map of the X chromosome covering 125 Mb of DNA in 25 contigs and containing 906 YAC clones . These contigs have been verified extensively by FISH and by gel and hybridization fingerprinting techniques . This independently derived map exceeds the coverage of recently reported X chromosome maps built as part of whole-genome YAC maps.

Biochem Mol Biol Int, 1996 Oct, 40(3), 611 - 6
Primary structures and sequence analysis of human ribosomal proteins L39 and S27; Tsui SK et al.; We report here the primary structures and sequence analysis of the human ribosomal protein L39 (hRPL39) and S27 (hRPS27) . The hRPL39 cDNA is 352 bp in size encoding a predicted protein of 51 amino acids . The region KRRHWRRTKL near the carboxyl end is conserved between human, rat, maize, C . elegans and yeast . The hRPS27 cDNA is 321 bp in size encoding a deduced protein of 84 amino acids . When the deduced amino acid sequence of hRPS27 was compared with that of rat ribosomal protein S27 and human metalloproteinstimulin-1 (MPS-1), identity levels of 96.4% and 100% were obtained respectively . A potential polyadenylation signal AACAAA is found in the MPS-1 cDNA but the more frequently used AATAAA sequence is present in the hRPS27 cDNA . The carboxyl-terminal cysteine arrangement in hRPS27 is similar to the family of C4 zinc finger DNA-binding proteins.

Cancer Genet Cytogenet, 1996 Oct 1, 91(1), 1 - 7
Amplification of a rearranged form of the high-mobility group protein gene HMGIC in OsA-CI osteosarcoma cells; Kools PF et al.; Recently the high-mobility group protein gene HMGIC has been found to be rearranged in a variety of benign mesenchymal tumors with 12q13-q15 aberrations, such as angiomyxoma, fibroadenomas of the breast, lipomas, pleomorphic salivary gland adenomas, polyps of the endometrium, pulmonary chondroid hamartomas, and uterine leiomyomas . Here we report on HMGIC aberrations in the osteosarcoma cell line OsA-CI . In Northern blot studies, aberrant HMGIC transcripts were detected . Analysis of cDNA sequence data obtained after 3' rapid amplification of cDNA ends, indicated these to consist of 5' HMGIC sequences encoding the three DNA binding domains fused to ectopic sequences apparently derived from part of the human lumican (keratan sulphate proteoglycan) gene (LUM), which we mapped by fluorescence in situ hybridization (FISH) to chromosome 12q22-q23 . Moreover, Southern blot analysis revealed amplification of this fusion gene but not of the 3'HMGIC sequences . This observation was independently confirmed by FISH analysis using yeast artificial chromosome (YAC) and cosmid clones, which furthermore indicated that the amplified 5'HMGIC sequences were contained within an amplicon of about 200 kb . Our results indicate that aberrations in HMGIC might not be restricted to benign mesenchymal tumors.

FASEB J, 1996 Oct, 10(12), 1378 - 87
The eIF-2alpha kinases and the control of protein synthesis; de Haro C et al.; Protein synthesis is regulated in response to environmental stimuli by covalent modification, primarily phosphorylation, of components of the translational machinery . Phosphorylation of the alpha subunit of eIF-2 is one of the best-characterized mechanisms for down-regulating protein synthesis in higher eukaryotes in response to various stress conditions . Three distinct protein kinases regulate protein synthesis in eukaryotic cells by phosphorylating the alpha subunit of eIF-2 at serine-51 . There are two mammalian eIF-2alpha kinases: the double-stranded RNA-dependent kinase (PKR) and heme-regulated inhibitor kinase (HRI), and the yeast GCN2 . The regulatory mechanisms and the molecular sizes of these eIF-2alpha kinases are different . The expression of PKR is induced by interferon, and the kinase activity is stimulated by low concentrations of double-stranded RNA . HRI is activated under heme-deficient conditions . Yeast GCN2 is activated by amino acid starvation . The phosphorylation of eIF-2alpha results in the shutdown of protein synthesis . Nevertheless, the eIF-2alpha kinases can regulate both global as well as specific mRNA translation . Inhibition of protein synthesis correlates with eIF-2alpha phosphorylation in response to a wide variety of different stimuli, including heat shock, serum deprivation, glucose starvation, amino acid starvation, exposure to heavy metal ions, and viral infection . Finally, recent studies suggest a role for eIF-2alpha phosphorylation in the control of cell growth and differentiation.

Development, 1996 Oct, 122(10), 2965 - 76
Discrete developmental stages during teliospore formation in the corn smut fungus, Ustilago maydis; Banuett F et al.; Ustilago maydis is a dimorphic fungus with a yeast-like non-pathogenic form and a filamentous (hyphal) pathogenic form that induces tumor formation in maize . Within mature tumors, hyphae give rise to teliospores, which are round, diploid cells surrounded by a specialized cell wall . Here we describe the time course of fungal development in the plant with a focus on the morphological changes in the hyphae and the pathway of teliospore formation . We confirm and extend earlier observations that U . maydis hyphae branch extensively on the leaf surface and intracellularly before induction of tumors . We observe that at later stages the filaments undergo a series of discrete morphogenetic changes leading to teliospore formation . In particular, we show that the hyphae become embedded in a mucilaginous matrix within the tumor cells and the hyphal tips become modified . The hyphae then undergo fragmentation to release individual cells that exhibit a variety of shapes on their way to becoming rounded . Finally, a specialized cell wall is deposited . Support for the existence of such a pathway comes from analysis of a mutant defective in the fuz1 gene: inactivation of fuz1 blocks production of the mucilaginous matrix and fragmentation of the hyphae, leading to a defect in teliospore formation . The different morphological changes that occur while in the plant but not in culture suggest that plant inputs play a key role in fungal development.

EMBO J, 1996 Oct 1, 15(19), 5370 - 82
Purification and biochemical heterogeneity of the mammalian SWI-SNF complex; Wang W et al.; We have purified distinct complexes of nine to 12 proteins {referred to as BRG1-associated factors (BAFs)} from several mammalian cell lines using an antibody to the SWI2-SNF2 homolog BRG1 . Microsequencing revealed that the 47 kDa BAF is identical to INI1 . Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene . A group of BAF47-associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co-purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI-SNF complex . Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4-VP16 to a nucleosomal template similar to the yeast SWI-SNF complex . Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition . The two SWI-SNF2 homologs, BRG1 and hbrm, were found in separate complexes . Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI-SNF complex may be tailored to the needs of a differentiated cell type.

EMBO J, 1996 Oct 1, 15(19), 5256 - 67
Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002; Brunn GJ et al.; The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR) . The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases . This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein, PHAS-1, in activated T lymphocytes . This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation . A mutant YAC-1 T lymphoma cell line, which was selected for resistance to the growth-inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation . In contrast, the PI 3-kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells . At similar drug concentrations (0.1-1 microM), wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR . The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor, LY294002, at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer . These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3-kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002.

Hum Mol Genet, 1996 Oct, 5(10), 1649 - 55
Characterization of the human ABC superfamily: isolation and mapping of 21 new genes using the expressed sequence tags database; Allikmets R et al.; As an approach to characterizing all human ATP-binding cassette (ABC) superfamily genes, a search of the human expressed sequence tag (EST) database was performed using sequences from known ABC genes . A total of 105 clones, containing sequences of potential ABC genes, were identified, representing 21 distinct genes . This brings the total number of characterized human ABC genes from 12 to 33 . The new ABC genes were mapped by PCR on somatic cell and radiation hybrid panels and yeast artificial chromosomes (YACs) . The genes are located on human chromosomes 1, 2, 3, 4, 6, 7, 10, 12, 13, 14, 16, 17 and X; at locations distinct from previously mapped members of the superfamily . The characterized genes display extensive diversity in sequence and expression pattern and this information was utilized to determine potential structural, functional and evolutionary relationships to previously characterized members of the ABC superfamily.

Hum Mol Genet, 1996 Oct, 5(10), 1589 - 98
cDNA cloning and chromosome mapping of the human Fe65 gene: interaction of the conserved cytoplasmic domains of the human beta-amyloid precursor protein and its homologues with the mouse Fe65 protein; Bressler SL et al.; Using the yeast two hybrid system, a mouse embryo cDNA library was screened for proteins that interact with the C-terminus of the human beta-amyloid precursor protein (beta PP) . A fusion protein was identified that interacts specifically with the cytoplasmic domain of beta PP and does not interact with the beta-amyloid region . The protein encoded by this partial mouse cDNA is identical to the C-terminus of the rat Fe65 protein . This mouse protein also interacts with the homologous C-terminal domains of the mouse amyloid precursor-like proteins, APLP1 and APLP2 . These conserved cytoplasmic regions contain a common amino acid motif, Asn-Pro-Thr-Tyr, which has previously been shown to influence both the secretion and internalization of beta PP . Fe65 has been implicated in regulatory and cell signaling mechanisms because it contains two different motifs involved in protein binding, a WW domain (a variant of Src homology 3 domains) and a phosphotyrosine interaction domain (PID) . Interestingly, the PID domain binds to the same motif present in the conserved cytoplasmic domains of the beta PP and beta PP-like proteins . RNA analyses reveal that Fe65 is predominantly expressed in brain and in the regions most affected by Alzheimer's disease (AD)-associated neuropathology . The human Fe65 mRNA was cloned from a fetal brain cDNA library . The message encodes a protein of 735 amino acids that is 95% identical to the rat Fe65 protein . The human Fe65 gene was mapped on human metaphase chromosomes to band 11p15 using fluorescence in situ hybridization.

Hum Mol Genet, 1996 Oct, 5(10), 1547 - 57
Positional cloning of a gene involved in hereditary multiple exostoses; Wuyts W et al.; Hereditary multiple exostosis (EXT) is an autosomal dominant condition mainly characterized by the presence of multiple exostoses on the long bones . These exostoses are benign cartilaginous tumors (enchondromata) . Three different EXT loci on chromosomes 8q (EXT1), 11p (EXT2) and 19p (EXT3) have been reported, and recently the EXT1 gene was identified by positional cloning . To isolate the EXT2 gene, we constructed a contig of yeast artificial chromosomes (YAC) and P1 clones covering the complete EXT2 candidate region on chromosome 11p11-p12 . One of the transcribed sequences isolated from this region corresponds to a novel gene with homology to the EXT1 gene, and harbours inactivating mutations in different patients with hereditary multiple exostoses . This indicates that this gene is the EXT2 gene . EXT2 has an open reading frame encoding 718 amino acids with an overall homology of 30.9% with EXT1, suggesting that a family of related genes might be responsible for the development of EXT.

Plant J, 1996 Oct, 10(4), 703 - 11
U-rich tracts enhance 3' splice site recognition in plant nuclei; Baynton CE et al.; The process of 5' and 3' splice site definition in plant pre-mRNA splicing differs from that in mammals and yeast . In mammals, splice sites are chosen by their complementarity to U1 snRNA surrounding the /GU at the 5' splice site and by the strength of the pyrimidine tract preceding the AG/ at the 3' splice site; in plants, the 3' intron boundary is defined in a position-dependent manner relative to AU-rich elements within the intron . To determine if uridines are utilized to any extent in plant 3' splice site recognition, uridines in the region preceding the normal (-1) 3' splice site of pea rbcS3A intron 1 were replaced with adenosines . This mutant activates two cryptic 3' splice sites (+62, +95) in the downstream exon, indicating that the uridines in the region immediately preceding the normal (-1) site are essential for recognition . Placement of different length uridine tracts upstream from the cryptic +62 site indicated that a cryptic exonic 3' splice site containing 14 or 10 uridine tracts with a G at -4 can effectively outcompete the normal 3' splice site containing an eight uridine tract with a U at -4 . Substitutions at the -4 position demonstrated that the identity of the nucleotide at this position greatly affects 3' splice site selection . It has been concluded that several factors affect competition between these 3' splice sites . These factors include the position of the AU transition point, the strength of the uridine tract immediately preceding the 3' terminal CAG/ and the identity of nucleotide -4.

Plant Physiol, 1996 Oct, 112(2), 767 - 77
Molecular and biochemical characterization of tomato farnesyl-protein transferase; Schmitt D et al.; The prenylation of membrane-associated proteins involved in the regulation of eukaryotic cell growth and signal transduction is critically important for their subcellular localization and biological activity . In contrast to mammalian cells and yeast, however, the function of protein prenylation in plants is not well understood and only a few prenylated proteins have been identified . We partially purified and characterized farnesyl-protein transferase from tomato (Lycopersicon esculentum, LeFTase) to analyze its biochemical and molecular properties . Using Ras- and G gamma-specific peptide substrates and competition assays we showed that tomato protein extracts have both farnesyl-protein transferase and geranylgeranyl-protein transferase 1 activities . Compared with the heterologous synthetic peptide substrates, the plant-specific CaaX sequence of the ANJ1 protein is a less efficient substrate for LeFTase in vitro . LeFTase activity profiles and LeFTase beta-subunit protein (LeFTB) levels differ significantly in various tissues and are regulated during fruit development . Partially purified LeFTase requires Zn2+ and Mg2+ for enzymatic activity and has an apparent molecular mass of 100 kD Immunoprecipitation experiments using anti-alpha LeFTB antibodies confirmed that LeFTB is a component of LeFTase but not of tomato geranylgeranyl-protein transferase 1 . Based on their conserved bio-chemical activities, we expect that prenyltransferases are likely integrated with the sterol biosynthesis pathway in the control of plant cell growth.

Nucleic Acids Res, 1996 Oct 1, 24(19), 3784 - 9
FLP-mediated recombination of FRT sites in the maize genome; Lyznik LA et al.; Molecular evidence is provided for genomic recombinations in maize cells induced by the yeast FLP/FRT site-specific recombination system . The FLP protein recombined FRT sites previously integrated into the maize genome leading to excision of a selectable marker, the neo gene . NPTII activity was not observed after the successful recombination process; instead, the gusA gene was activated by the removal of the blocking DNA fragment . Genomic sequencing in the region of the FRT site (following the recombination reaction) indicated that a precise rearrangement of genomic DNA sequences had taken place . The functional FLP gene could be either expressed transiently or after stable integration into the maize genome . The efficiency of genomic recombinations was high enough that a selection for recombination products, or for FLP expression, was not required . The results presented here establish the FLP/FRT site-specific recombination system as an important tool for controlled modifications of maize genomic DNA.

Biochem J, 1996 Oct 1, 319 ( Pt 1), 109 - 16
Reconstitution of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes: analysis of protein X involvement and interaction of homologous and heterologous dihydrolipoamide dehydrogenases; Sanderson SJ et al.; Optimal conditions for rapid and efficient reconstitution of pyruvate dehydrogenase complex (PDC) activity are demonstrated by using an improved method for the dissociation of the multienzyme complex into its constituent E1 (substrate-specific 2-oxoacid decarboxylase) and E3 (dihydrolipoamide dehydrogenase) components and isolated E2/X (where E2 is dihydrolipoamide acyltransferase) core assembly . Selective cleavage of the protein X component of the purified E2/X core with the proteinase arg C decreases the activity of the reconstituted complex to residual levels (i.e . 8-12%); however, significant recovery of reconstitution is achieved on addition of a large excess (i.e . 50-fold) of parent E3 . N-terminal sequence analysis of the truncated 35,000-M(r) protein X fragment locates the site of cleavage by arg C at the extreme N-terminal boundary of a putative E3-binding domain and corresponds to the release of a 15,000-M(r) N-terminal fragment comprising both the lipoyl and linker sequences . In native PDC this region of protein X is shown to be partly protected from proteolytic attack by the presence of E3 . Recovery of complex activity in the presence of excess E3 after arg C treatment is thought to result from low-affinity interactions with the partly disrupted subunit-binding domain on X and/or the intact analogous subunit binding domain on E2 . Contrasting recoveries for arg C-modified E2/X/E1 core, and untreated E2/E1 core of the 2-oxoglutarate dehydrogenase complex, reconstituted with excess bovine heart E3, pig heart E3 or yeast E3 point to subtle differences in subunit interactions with heterologous E3s and offer an explanation for the inability of previous investigators to achieve restoration of PDC function after selective proteolysis of the protein X component.

Mol Pharmacol, 1996 Oct, 50(4), 891 - 9
Mapping the functional domains of human recombinant phosphodiesterase 4A: structural requirements for catalytic activity and rolipram binding; Jacobitz S et al.; To identify functional domains of the 886-amino acid human recombinant cAMP-specific phosphodiesterase (PDE) subtype A (rhPDE4A), we engineered the expression of seven mutant proteins containing both NH2- and COOH-terminal truncations . The level of rhPDE4A protein expression in yeast was monitored by immunoblotting using enzyme-specific antisera . Biochemical profiles of the mutant proteins were compared with those of the full-length protein or a fully active truncated form of the enzyme (rhPDE4A Met265-886), lacking the first 264 amino acids . The smallest catalytically active fragment generated was Met332-722, which at 45 kDa is less than half the mass of the full-length enzyme (approximately 110 kDa) but spans the most highly conserved region of the PDE superfamily . Two prototypical PDE4 inhibitors, rolipram and RP 73401, inhibited cAMP hydrolyzing activity of all truncated forms of the enzyme, with IC50 values of 70-2000 nM and 0.2-0.6 nM, respectively . {3H}(R)-Rolipram bound to two sites on Met265-886, a high affinity site (Kd1 = 0.7 +/- 0.3 nM) and a low affinity site (Kd2 = 34 +/- 10 nM) . Interestingly, {3H}(R)-rolipram failed to bind to Met332-886 with high affinity, indicating that high affinity binding is not required for inhibition of enzyme activity . Low affinity rolipram binding was still present in Met332-886 (Kd = 101 +/- 7 nM) . In contrast to {3H}(R)-rolipram, {3H}RP 73401 bound to a single class of high affinity sites on Met265-886 (Kd = 0.4 +/- 0.1 nM) . Further truncation of the enzyme to Met332-886 had no effect on {3H}RP 73401 binding (Kd = 0.2 +/- 0.03 nM) . We conclude that the catalytic center of rhPDE4A lies between amino acids 332 and 722 . Furthermore, amino acids 265-332 may form a high affinity binding site for rolipram that is outside of the catalytic domain . As a more likely alternative, these amino acids may not form a distinct binding site but instead may be required for the recombinant enzyme to assume a conformation that binds rolipram at the catalytic domain with a high affinity.

Am J Pathol, 1996 Oct, 149(4), 1221 - 8
Germ cell tumors of the testis overexpress wild-type p53; Guillou L et al.; Several recent studies have suggested that testicular germ cell tumors express high levels of wild-type p53 protein . To clarify and confirm this unexpected result, we have investigated seminomatous and nonseminomatous germ cell tumors at the genomic, mRNA, and protein levels . Thirty-five tumors were examined for p53 overexpression using antibodies directed against the p53 (PAb1801, PAb240, and CM1), mdm2 (IF2), and p21Waf1/Clp1 (EA10) proteins . Thirty-two tumors were screened for p53 mutations by single-strand conformation polymorphism analysis . Eighteen tumors were screened with a functional assay that tests the transcriptional competence of human p53 protein expressed in yeast . On frozen sections, 100, 65, 35, 73, and 0% of tumors reacted with the CM1, PAb240, PAb1801, IF2, and EA10 antibodies, respectively . No p53 mutations were detected by single-strand conformation polymorphism or by functional assay . The fact that many tumors overexpress wild-type p53 but not mdm2 rules out mdm2 overexpression as a general explanation for the presence of wild-type p53 in these tumors . The absence of p21 overexpression suggests that p53 may be unable to activate transcription of critical target genes, which may explain why the presence of wild-type p53 is tolerated in this tumor type, although the mechanism for this transcriptional inactivity remains to be established.

J Neurochem, 1996 Oct, 67(4), 1744 - 50
Accelerated myelinogenesis by dietary lipids in rat brain; Salvati S et al.; Our previous work showed an early development of behavioral reflexes in rats whose mothers had been fed, during pregnancy and lactation, a lipid fraction extracted from yeast grown on n-alkanes (which contain 50% odd-chain fatty acids) in comparison with controls fed a margarine diet . To clarify whether the observed changes might be linked to an early myelination, we have investigated mRNAs involved in myelin synthesis in the brains of offspring at 5 days of age by northern blot and in situ hybridization . Northern blot analysis showed that proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) mRNAs were higher in animals on the lipid diet compared with controls . In situ hybridization with probes specific for PLP, myelin basic protein, and MOG mRNA showed significantly higher numbers of positive cells in test animals compared with controls in all brain regions . This study shows an acceleration of myelinogenesis induced by dietary lipids . These data can give a new insight in the therapeutical approaches involved to promote repair in demyelinating diseases.

J Cell Biol, 1996 Oct, 135(1), 85 - 95
Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor; Gould SJ et al.; Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome . We have identified Pex13p, a novel integral peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain . Although only a small amount of Pex5p is bound to peroxisomes at steady state (< 5%), loss of Pex13p further reduces the amount of peroxisome-associated Pex5p by approximately 40-fold . Furthermore, loss of Pex13p eliminates import of peroxisomal matrix proteins that contain either the type-1 or type-2 peroxisomal targeting signal but does not affect targeting and insertion of integral peroxisomal membrane proteins . We conclude that Pex13p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor.

J Cell Biol, 1996 Oct, 135(1), 53 - 61
Architecture of coatomer: molecular characterization of delta-COP and protein interactions within the complex; Faulstich D et al.; Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles . In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, {COPs} alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits . We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing . delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2 . We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes . A knock-out of the delta-COP gene in yeast is lethal . Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs . Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs . We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.

Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 11274 - 9
Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought; Jonak C et al.; Yeast and animals use mitogen-activated protein (MAP) kinase cascades to mediate stress and extracellular signals . We have tested whether MAP kinases are involved in mediating environmental stress responses in plants . Using specific peptide antibodies that were raised against different alfalfa MAP kinases, we found exclusive activation of p44MMK4 kinase in drought- and cold-treated plants . p44MMK4 kinase was transiently activated by these treatments and was correlated with a shift in the electrophoretic mobility of the p44MMK4 protein . Although transcript levels of the MMK4 gene accumulated after drought and cold treatment, no changes in p44MMK4 steady state protein levels were observed, indicating a posttranslational activation mechanism . Extreme temperatures, drought, and salt stress are considered to be different forms of osmotic stress . However, high salt concentrations or heat shock did not induce activation of p44MMK4, indicating the existence of distinct mechanisms to mediate different stresses in alfalfa . Stress adaptation in plants is mediated by abscisic acid (ABA)-dependent and ABA-independent processes . Although ABA rapidly induced the transcription of an ABA-inducible marker gene, MMK4 transcript levels did not increase and p44MMK4 kinase was not activated . These data indicate that the MMK4 kinase pathway mediates drought and cold signaling independently of ABA.

Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10923 - 7
RIP and FADD: two "death domain"-containing proteins can induce apoptosis by convergent, but dissociable, pathways; Grimm S et al.; With use of the yeast two-hybrid system, the proteins RIP and FADD/MORT1 have been shown to interact with the "death domain" of the Fas receptor . Both of these proteins induce apoptosis in mammalian cells . Using receptor fusion constructs, we provide evidence that the self-association of the death domain of RIP by itself is sufficient to elicit apoptosis . However, both the death domain and the adjacent alpha-helical region of RIP are required for the optimal cell killing induced by the overexpression of this gene . By contrast, FADD's ability to induce cell death does not depend on crosslinking . Furthermore, RIP and FADD appear to activate different apoptotic pathways since RIP is able to induce cell death in a cell line that is resistant to the apoptotic effects of Fas, tumor necrosis factor, and FADD . Consistent with this, a dominant negative mutant of FADD, lacking its N-terminal domain, blocks apoptosis induced by RIP but not by FADD . Since both pathways are blocked by CrmA, the interleukin 1 beta converting enzyme family protease inhibitor, these results suggest that FADD and RIP can act along separable pathways that nonetheless converge on a member of the interleukin 1 beta converting enzyme family of cysteine proteases.

Mamm Genome, 1996 Oct, 7(10), 758 - 66
A radiation hybrid map spanning the entire human X chromosome integrating YACs, genes, and STS markers; Kumlien J et al.; We present a radiation hybrid (RH) map of human Chromosome (Chr) X, using 50 markers on 72 radiation hybrids . The markers, obtained from the consensus map, form a grid spanning the entire chromosome . To check the RH map, the marker order was determined by analysis of presence or absence of retained human DNA fragments in the RHs; the comparison with the consensus showed a similar order . Any STSs, microsatellites, genes, and clones can be positioned and ordered relative to the marker grid . This approach integrates genetic, physical, and large-scale clone mapping and is used to link YAC contigs containing data from various experimental sources.

J Infect Dis, 1996 Oct, 174(4), 862 - 6
Immunization with recombinant p17/p24:Ty virus-like particles in human immunodeficiency virus-infected persons; Veenstra J et al.; In studies of the natural history of human immunodeficiency virus type 1 (HIV-1) infection, it has been repeatedly shown that higher-titer antibody responses to the HIV gag p24 protein correlate with less rapid disease progression . In HIV-negative persons, immunization with HIV-1 p17/p24:Ty virus-like particles (p24-VLP) induced humoral and cellular immune responses to p24 . This construct was therefore studied as a potential immunotherapeutic agent with the objective of augmenting the immune response to p24 in a double-blind placebo-controlled trial involving 74 p24 antibody-positive, asymptomatic HIV-1-infected subjects with CD4 cell counts > 350/mm3 . Immunization with p24-VLP was generally well tolerated . Immunization with p24-VLP did not increase p24 antibody levels and had no effect on CD4 cell counts or virus load . The failure to increase p24 antibody titers cannot entirely be explained by the subjects' immunodeficiency because most generated an antibody response to Ty, a yeast component of the immunogen.

Arch Biochem Biophys, 1996 Oct 1, 334(1), 104 - 12
Isolation and characterization of novel long-chain acyl-CoA thioesterase/carboxylesterase isoenzymes from Candida rugosa; Diczfalusy MA et al.; Long-chain acyl-CoA thioesterases, which catalyze the cleavage of acyl-CoA's to free fatty acids and CoASH, are abundant in animal cells . However, in yeast little is known about presence and function of acyl-CoA thioesterase activity . Therefore a commercial lipase preparation from the yeast Candida rugosa was investigated and found to contain high myristoyl-CoA thioesterase activity . Hydrophobic interaction chromatography separated the activity into three peaks, of which two enzymes (YTE-1 and YTE-2) were purified to apparent homogeneity with molecular masses of about 40 kDa as determined by size-exclusion chromatography and SDS-PAGE . The employed purification protocol resulted in final preparations with specific activities of about 90 micromol/mg/min with myristoyl-CoA as substrate . YTE-1 and YTE-2 showed similar kinetic properties and YTE-1 was characterized in detail . Acyl-CoA chain-length specificity showed that YTE-1 was not active on acyl-CoAs shorter than decanoyl-CoA, at the substrate concentrations tested . The best substrates were C14-C18 acyl-CoAs with Vmax values of about 150 micromol/mg/min and Km values of 15-46 microM . The enzyme was very active with lauroyl-CoA (Vmax about 400 micromol/mg/min) although the Km was high (about 325 microM) . The purified enzyme was also active on short-chain nitrophenyl esters but inactive with tributyrin . Treatment of the protein with N-glycosidase F decreased the molecular mass about 1-2 kDa, indicating the presence of carbohydrate of the high mannose type . Diisopropyl fluorophosphate (DFP) inhibited the enzyme activity efficiently and the protein was covalently labeled with {3H}DFP . p-Chloromercuribenzoic acid inhibited the thioesterase activity but did not affect carboxylesterase activity . N-terminal sequence analysis and labeling by DFP suggest that these long-chain acyl-CoA thioesterases belong to a novel group of yeast serine esterases.

Cancer Res, 1996 Oct 1, 56(19), 4493 - 8
Early involvement of 6q in surface epithelial ovarian tumors; Tibiletti MG et al.; Complex karyotypes are often seen in primary surface epithelial ovarian tumors (SEOTs) . Conventional cytogenetic as well as fluorescence in situ hybridization analyses coupled with loss of heterozygosity studies identified abnormalities of chromosome 6 as one of the most frequent lesions in these types of tumors . We performed cytogenetic analysis of direct preparations from 40 SEOTs, including borderline tumors and low-, intermediate-, and high-grade carcinomas to verify the frequency of chromosome 6 alterations . We also carried out fluorescence in situ hybridization analysis with a chromosome 6 library and yeast artificial chromosome clones from a region of the same chromosome (6q27) . Chromosome 6 abnormalities were identified in 30 of 32 analyzable SEOTs . Twenty-five of 32 cases showed a deletion of 6q irrespective of their histological grade . We wish to underline that this is the first report proving that del(6q) was the most frequent chromosome anomaly in near-diploid SEOTs and that it was the sole anomaly observed in four SEOTs with diploid complement . Our findings suggest that abnormalities of the telomeric region of chromosome 6 (6q27) may be considered one of the earliest lesions in the pathogenesis of ovarian carcinomas.

J Mol Evol, 1996 Oct, 43(4), 384 - 98
Molecular evolution of the 14-3-3 protein family; Wang W et al.; Members of the highly conserved and ubiquitous 14-3-3 protein family modulate a wide variety of cellular processes . To determine the evolutionary relationships among specific 14-3-3 proteins in different plant, animal, and fungal species and to initiate a predictive analysis of isoform-specific differences in light of the latest functional and structural studies of 14-3-3, multiple alignments were constructed from forty-six 14-3-3 sequences retrieved from the GenBank and SwissProt databases and a newly identified second 14-3-3 gene from Caenorhabditis elegans . The alignment revealed five highly conserved sequence blocks . Blocks 2-5 correlate well with the alpha helices 3, 5, 7, and 9 which form the proposed internal binding domain in the three-dimensional structure model of the functioning dimer . Amino acid differences within the functional and structural domains of plant and animal 14-3-3 proteins were identified which may account for functional diversity amongst isoforms . Protein phylogenic trees were constructed using both the maximum parsimony and neighbor joining methods of the PHYLIP(3.5c) package; 14-3-3 proteins from Entamoeba histolytica, an amitochondrial protozoa, were employed as an outgroup in our analysis . Epsilon isoforms from the animal lineage form a distinct grouping in both trees, which suggests an early divergence from the other animal isoforms . Epsilons were found to be more similar to yeast and plant isoforms than other animal isoforms at numerous amino acid positions, and thus epsilon may have retained functional characteristics of the ancestral protein . The known invertebrate proteins group with the nonepsilon mammalian isoforms . Most of the current 14-3-3 isoform diversity probably arose through independent duplication events after the divergence of the major eukaryotic kingdoms . Divergence of the seven mammalian isoforms beta, zeta, gamma, eta, epsilon, tau, and sigma (stratifin/HME1) occurred before the divergence of mammalian and perhaps before the divergence of vertebrate species . A possible ancestral 14-3-3 sequence is proposed.

Hum Genet, 1996 Oct, 98(4), 497 - 9
The gene encoding the p60 subunit of chromatin assembly factor I (CAF1P60) maps to human chromosome 21q22.2, a region associated with some of the major features of Down syndrome; Katsanis N et al.; Trisomy 21 is the most common aneuploidy in humans with a frequency of 1 in 700 live births and is by far the most common defined cause of mental retardation . To analyse which of the chromosome 21 genes is overexpressed early in development-giving rise to the Down syndrome phenotype-and to provide candidate genes for other HSA21 disease loci, we need a transcription map of the chromosome . Therefore, to enrich the gene map of human chromosome 21 we have undertaken a systematic approach to fine mapping and characterising expressed sequences generated by the various cDNA sequencing projects . In this report we show the localisation of the CAF1P60 gene to human chromosome 21 and its fine mapping to 21q22.2 between D21S333 and D21S334 . This mapping position places CAF1P60 in a region of HSA21 which is strongly associated with the major features of Down syndrome . The function of this gene product may have important implications for the phenotype that arises from trisomy 21.

Hum Genet, 1996 Oct, 98(4), 460 - 6
YAC and cosmid FISH mapping of an unbalanced chromosomal translocation causing partial trisomy 21 and Down syndrome; Nadal M et al.; Most cases of Down syndrome (DS) result from a supernumerary chromosome 21; however, there are rare cases in which DS is due to partial trisomy of chromosome 21, involving various segments of the chromosome . The characterization of cases of DS that are due to partial trisomy 21 allows the phenotype to be correlated with the genotype . We present a case with features of DS and a partial trisomy of chromosome 21 inherited from a paternal balanced translocation involving chromosomes 13 and 21 . Fluorescence in situ hybridization analysis using yeast artificial chromosome (YAC) probes mapped the breakpoint to 21q22.1, within YAC 230E8, which contains markers CBR, D21S333 and D21S334 . Further mapping using cosmids positioned the breakpoint proximal to CBR . The patient was also monosomic for the distal portion of chromosome 13 (q33-qter) . Many phenotypic features of DS were present including hypotonia, flat occiput, flat facies, up-slanted palpebral fissures, epicanthic folds, flat nasal bridge, macroglossia, open mouth, small ears and a heart murmur . This case further supports the contention that the majority of the phenotypic features of DS map to 21q22-qter and further refines the location of some of them . In addition to the DS phenotype, the patient had a prominent upper maxilla with protruding upper incisors, and low levels of the coagulation factors VII and X, consistent with a syndrome resulting from monosomy 13q33-qter . Since some features overlap between the two syndromes, including severe mental retardation, it is unclear to what extent monosmy for 13q33-qter, trisomy for 21q22.1-qter, or a combination of both, contributed to the common features of the phenotype.

Hum Genet, 1996 Oct, 98(4), 454 - 9
A detailed investigation of two cases exhibiting characteristics of the 6p deletion syndrome; Davies AF et al.; Deletions of the short arm of chromosome 6 are relatively rare, only 16 cases having been described in the literature so far . Here we present a detailed investigation by fluorescence in situ hybridisation of two further cases with different but overlapping interstitial deletions involving 6p22, 6p23 and 6p24 . The main features involved are craniofacial malformations, heart and kidney defects, mental retardation/developmental delay, hypotonia and hydrocephalus . By using 36 yeast artificial chromosome and cosmid clones from a contig covering 6p22.3-6p25 and other probes with defined cytogenetic locations within 6p21-6p22 we have precisely localised the breakpoints involved in each of the cases, estimated the sizes of the deleted regions and defined the region that is hemizygously deleted in both cases.

Hum Genet, 1996 Oct, 98(4), 443 - 6
Physical linkage and orientation of the human complement C8 alpha and C8 beta genes on chromosome 1p32; Platteborze PL et al.; Human C8 is one of five components (C5b, C6, C7, C8, C9) of the cytolytic C5b-9 complex of complement . It consists of three nonidentical subunits (C8 alpha, C8 beta, C8 gamma), which are encoded in separate genes . Genetic linkage and chromosomal localization studies previously established that C8 alpha and C8 beta are closely linked on chromosome 1p32 . In this study, clones with inserts containing genes for both C8 alpha and C8 beta were isolated from a yeast artificial chromosome (YAC) human genomic DNA library and characterized in an effort to determine intergenic distance and orientation . One clone with a approximately 330-kb insert yielded restriction digest patterns for C8 alpha and C8 beta that agreed with those obtained previously from digests of human genomic DNA, thereby confirming the presence of intact copies of both genes . A second clone with a approximately 280-kb insert yielded similar results; however, it was truncated at the 5' end of the C8 alpha gene . Restriction digests of both clones were subjected to PFGE and Southern blot analysis using probes specific for the terminal exons of C8 alpha (exons 1 and 11) and C8 beta (exons 1 and 12) . Results indicate the genes are physically linked (< 23 kb) and in a 3'-3' orientation . This is the same orientation as the ancestrally related C6 and C7 genes, which are also physically linked on chromosome 5p13.

Atherosclerosis, 1996 Sep 27, 126(1), 65 - 75
Inhibition of LDL oxidation and myeloperoxidase dependent tyrosyl radical formation by the selective estrogen receptor modulator raloxifene (LY139481 HCL); Zuckerman SH et al.; Cellular oxidation of protein and lipoproteins is believed to contribute to the pathology associated with both acute and chronic inflammatory processes . Enzymatic, myeloperoxidase and lipoxygenase, and non- enzymatic oxidation of low density lipoprotein, LDL, has been implicated in foam cell formation and the progression of atherosclerotic changes within the arterial wall . In the present study, the in vitro protective role of the selective estrogen receptor modulator, raloxifene, in these oxidant triggered processes has been investigated . Raloxifene, as with estrogen was observed to inhibit both copper mediated LDL oxidation as well as the cellular modification of LDL by murine peritoneal macrophages . Raloxifene was, however, a more potent inhibitor of LDL oxidation than 17 beta-estradiol . The inhibition of macrophage LDL modification by raloxifene was not due to a non-specific effect on all effector functions as phagocytosis of opsonized yeast was comparable with control macrophage cultures . In addition to the protective effects on LDL oxidation, raloxifene also inhibited tyrosyl radical formation catalyzed by myeloperoxidase . The inhibition of myeloperoxidase activity was observed for both the isolated enzyme and in phorbol ester stimulated murine peritoneal neutrophils . In contrast, raloxifene was a weaker inhibitor of horseradish peroxidase . These results demonstrate a potential protective role for raloxifene as an anti-oxidant in in vitro assays designed to evaluate oxidant mediated radical formation and tissue damage.

J Biol Chem, 1996 Sep 27, 271(39), 24048 - 54
Endosomal localization of the autoantigen EEA1 is mediated by a zinc-binding FYVE finger; Stenmark H et al.; EEA1, a 162-kDa autoantigen associated with subacute cutaneous systemic lupus erythematosus, is a coiled-coil protein localized to early endosomes and cytosol . At its C terminus, the protein contains a cysteine-rich motif, which is shared with Vps27, Fab1, and Vac1, yeast proteins implicated in membrane traffic (Mu, F . T., Callaghan, J . M., Steele-Mortimer, O., Stenmark, H., Parton, R . G., Campbell, P . L., McCluskey, J., Yeo, J . P., Tock, E . P., and Toh, B . H . (1995) J . Biol . Chem . 270, 13503-13511) . Here we show that this motif constitutes a genuine zinc binding domain, which we term the FYVE finger (based on the first letters of four proteins containing this motif) . Profile-based data base searches identified the FYVE finger in 11 distinct proteins . The FYVE finger-containing C terminus of EEA1 was found to bind 2 mol equivalents of Zn2+ . Mutations of conserved histidine and cysteine residues in the FYVE motif independently reduced zinc binding to 1 mol equivalent . Confocal immunofluorescence microscopy of transfected HEp2 cells revealed that the C-terminal part (residues 1277-1411) of EEA1 colocalizes extensively with a GTPase-deficient mutant of the early endosomal GTPase Rab5, while deletion of the FYVE finger or mutations that interfere with zinc binding cause a cytosolic localization . These results implicate the FYVE finger in the specific localization of EEA1 to endosomes.

J Biol Chem, 1996 Sep 27, 271(39), 23653 - 6
Identification of the GalNAc kinase amino acid sequence; Pastuszak I et al.; A new kinase that forms GalNAc-1-P was purified from pig kidney cytosol and identified on gels by labeling with N3-{32P}ATP (Pastuszak, I., Drake, R., and Elbein, A . D . (1996) J . Biol . Chem . 271, in press) . A 50-kDa labeled protein was eluted, digested with trypsin, and the sequences of four peptides representing 49 amino acids showed 90% identity to sequence of human galactokinase reported to be on chromosome 15 . To resolve this dilemma, activities and substrate specificities of galactokinase and GalNAc kinase from human and pig kidney, as well as of galactokinase from the yeast clone transfected with the cDNA from presumptive human galactokinase, were compared . The purified galactokinases phosphorylated galactose, but not GalNAc, whereas GalNAc kinase also phosphorylated galactose when this sugar was present at millimolar concentrations . Extracts of gal 1(-) yeast clone, transfected with presumptive human galactokinase cDNA, had very low galactokinase activity even when yeast were grown on galactose, but good activity with GalNAc . On the other hand, the wild type yeast phosphorylated galactose, but not GalNAc . These data indicate that the sequence reported for galactokinase on chromosome 15 is that of GalNAc kinase, which can phosphorylate galactose when this sugar is present at millimolar concentrations . This transfection thus allows the yeast mutant to grow slowly on galactose-containing media.

Mol Gen Genet, 1996 Sep 25, 252(4), 483 - 8
Characterization and application of soybean YACs to molecular cytogenetics; Zhu T et al.; Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes . In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH) . We have screened such a library for low-copy-number clones by hybridization to total genomic DNA . Four clones were chosen for chromosome tagging based upon their low or moderate signal . By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei . FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci . The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes . We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.

J Biol Chem, 1996 Sep 20, 271(38), 23022 - 8
Identification of the Rho-binding domain of p160ROCK, a Rho-associated coiled-coil containing protein kinase; Fujisawa K et al.; A protein serine/threonine kinase, p160(ROCK), has been identified as a putative Rho target protein that is activated when bound to the GTP-bound form of the small GTPase Rho (Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatu, A., Fujita, A., Watanabe, N . Saito, Y., Kakizuka, A., Morii, N., and Narumiya, S . (1996) EMBO J . 15, 1885-1893) . p160(ROCK) has a serine/threonine kinase domain in its NH2-terminal region, followed by an approximately 600-amino acid-long alpha-helix, a cysteine-rich zinc finger-like motif, and a pleckstrin homology region in the COOH terminus . To identify the Rho binding domain of this protein, we divided p160 into five fragments, expressed each as a His-tagged recombinant protein, and performed a ligand overlay assay using {35S}guanosine-5'-3-O-(thio)triphosphate (GTPgammaS)-bound glutathione S-transferase-RhoA . Specific GTPgammaS-Rho binding was observed only in the fragment M2, which covered most of the carboxyl half of the alpha-helix between amino acids 727 and 1021 . This fragment was further subdivided into several fragments, and the ligand overlay assay as well as the yeast two hybrid system was carried out to identify the Rho-binding region . These studies localized the minimum Rho binding region to amino acids 934-1015 . To identify critical amino acids for Rho binding, we analyzed the Rho binding activity of the subfragment with various point mutations . This analysis revealed that K934M, L941A, and E1008A mutations significantly weakened Rho binding and an I1009A mutation abolished Rho binding . The amino acid sequence in this region had no significant homology with Rho effector motif class 1, which is shared by putative Rho targets, PKN, rhophilin, and rhotekin, (Reid, T., Furuyashiki, T., Ishizaki, T., Watanabe, G., Watanabe, N., Fujisawa, K., Morii, N., Madaule, P., and Narumiya, S . (1996) J . Biol . Chem . 271, 13556-13560) and may define a distinct class of Rho effector motif.

J Biol Chem, 1996 Sep 20, 271(38), 22941 - 4
FKBP-12 recognition is dispensable for signal generation by type I transforming growth factor-beta receptors; Charng MJ et al.; The FK506-binding protein, FKBP12, is a putative target of type I receptors for transforming growth factor-beta (TbetaR-I) . As the FK506 motif that competes with TbetaR-I for FKBP12 resembles an invariant Leu-Pro dipeptide in TbetaR-I, we replaced Leu193 and Pro194 with Ala, along with mutations across the Gly/Ser box . L193A, P194A, and L193A/P194A do not alter TbetaR-I function; T204D partially activates, independent of ligand; L193A/P194A/T204D was an even more potent constitutive mutation . Association with FKBP12 in a yeast two-hybrid assay was disrupted by P194A, L193A/P194A, and L193A/P194A/T204D, but not L193A or T204D alone . Thus, FKBP12 recognition is dispensable for TGFbeta signaling.

EMBO J, 1996 Sep 16, 15(18), 4909 - 18
Association of inhibitory tyrosine protein kinase p50csk with protein tyrosine phosphatase PEP in T cells and other hemopoietic cells; Cloutier JF et al.; p50csk is a tyrosine protein kinase (TPK) that represses the activity of Src family TPKs . We previously showed that Csk is a potent negative regulator of antigen receptor signaling in T lymphocytes and that its Src homology (SH) 3 and SH2 domains are required to inhibit these signals . To test the idea that the Csk SH3 and SH2 domains mediate interactions with other cellular proteins, we attempted to identify Csk-associated polypeptides using the yeast two-hybrid system . The results of our experiments demonstrated that Csk physically associates with PEP, a protein tyrosine phosphatase (PTP) expressed in hemopoietic cells . Further analyses revealed that this interaction was mediated by the Csk SH3 domain and by a proline-rich region (PPPLPERTP) in the non-catalytic C-terminal portion of PEP . The association between Csk and PEP was documented in transiently transfected Cos-1 cells and in a variety of cells of hemopoietic lineages, including T cells . Additional analyses demonstrated that the association between Csk and PEP is highly specific . Together, these data indicated that PEP may be an effector and/or a regulator of p50csk in T cells and other hemopoietic cells . Moreover, they allowed the identification of PEP as the first known ligand for the Csk SH3 domain.

Genomics, 1996 Sep 15, 36(3), 522 - 6
The genes encoding the human CC-chemokine receptors CC-CKR1 to CC-CKR5 (CMKBR1-CMKBR5) are clustered in the p21.3-p24 region of chromosome 3; Samson M et al.; The five human CC-chemokine receptors functionally characterized to date were mapped by using a radiation hybrid panel and YAC contigs . The genes encoding CC-CKR1, CC-CKR2, CC-CKR3, and CC-CKR5 (designated respectively CMKBR1, CMKBR2, CMKBR3, and CMKBR5 in the Genome Data Bank) were found to be clustered in the 3p21.3 region of chromosome 3, between the AFM362WB9 and the WI-6983 markers . The four genes fall within a total distance of about 350 kb . The fifth gene (CMKBR4, encoding the CC-CKR4 receptor) was located more distally (3p24) on the same chromosome, between the FB18G7 and the D3S1768 markers . These localizations were confirmed by mapping the genes into the YAC contigs covering these regions . The clustering of chemokine receptor genes suggests a relatively recent expansion of the gene family by gene duplication . Deletions and duplications of the 3p21 region have been described in neoplastic disorders of the hematopoietic lineage, suggesting a potential link with the CC-chemokine receptor gene family.

Genomics, 1996 Sep 15, 36(3), 507 - 14
Refinement of the Van der Woude gene location and construction of a 3.5-Mb YAC contig and STS map spanning the critical region in 1q32-q41; Schutte BC et al.; Van der Woude syndrome (VWS) is the most frequent form of syndromic clefting . Linkage analysis has localized the gene between D1S245 and D1S414, an interval of 4.1 cM with the following order of loci: centromere-D1S245/D1S471-D1S491-D1S205-D1S414 -telomere . A microdeletion around D1S205 aided in narrowing the critical region to D1S491-D1S414 by heterozygosity testing . In this study, the location was refined by detection of a recombinant with D1S205 in a new family, indicating that VWS lies between D1S491 and D1S205, a 1.6-cM interval . A roughly 3.5-Mb YAC contig was built from D1S245 through D1S414, encompassing the interval D1S491-D1S205 in level 1 or level 2 paths . Clones were assembled by sequence tagged site (STS) content using the five polymorphic markers from above, four novel STSs identified from YAC ends, and a new STS derived from probe CRI-L461 (D1S70) . D1S70 was assigned to the critical region . One single YAC, yCEPH785B2, contains both flanking STSs (D1S491, D1S205) . STS content mapping suggests neither chimerism nor deletion of yCEPH785B2 but does suggest that the maximum size of the critical region is approximately 850 kb . All STSs were tested for their presence on a somatic cell hybrid containing the microdeleted chromosome 1 as the sole human chromosome 1 component . Both the proximal and distal ends of the microdeletion mapped to the 850-kb YAC, yCEPH785B2 . Therefore, the microdeletion overlapped the critical region, confirming the genetic recombinant data.

Genomics, 1996 Sep 15, 36(3), 492 - 506
Mapping human telomere regions with YAC and P1 clones: chromosome-specific markers for 27 telomeres including 149 STSs and 24 polymorphisms for 14 proterminal regions; Vocero-Akbani A et al.; A YAC library enriched for telomere clones was constructed and screened for the human telomere-specific repeat sequence (TTAGGG) . Altogether 196 TYAC library clones were studied: 189 new TYAC clones were isolated, 149 STSs were developed for 132 different TY-ACs, and 39 P1 clones were identified using 19 STSs from 16 of the TYACs . A combination of mapping methods including fluorescence in situ hybridization, somatic cell hybrid panels, clamped homogeneous electric fields, meiotic linkage, and BLASTN sequence analysis was utilized to characterize the resource . Forty-five of the TYACs map to 31 specific telomere regions . Twenty-four linkage markers were developed and mapped within 14 proterminal regions (12 telomeres and 2 terminal bands) . The polymorphic markers include 12 microsatellites for 10 telomeres (1q, 2p, 6q, 7q, 10p, 10q, 13q, 14q, 18p, 22q) and the terminal bands of 11q and 12p . Twelve RFLP markers were identified and meiotically mapped to the telomeres of 2q, 7q, 8p, and 14q . Chromosome-specific STSs for 27 telomeres were identified from the 196 TYACs . More than 30,000 nucleotides derived from the TYAC vector-insert junction regions or from regions flanking TYAC microsatellites were compared to reported sequences using BLASTN . In addition to identifying homology with previously reported telomere sequences and human repeat elements, gene sequences and a number of ESTs were found to be highly homologous to the TYAC sequences . These genes include human coagulation factor V (F5), Weel protein tyrosine kinase (WEE1), neurotropic protein tyrosine kinase type 2 (NTRE2), glutathione S-transferase (GST1), and beta tubulin (TUBB) . The TYAC/P1 resource, derivative STSs, and polymorphisms constitute an enabling resource to further studies of telomere structure and function and a means for physical and genetic map integration and closure.

Genomics, 1996 Sep 15, 36(3), 459 - 67
Direct complementation of Chlamydomonas mutants with amplified YAC DNA; Vashishtha M et al.; We previously described the construction and characterization of a Chlamydomonas genomic library in yeast artificial chromosomes (YACs) . Here we describe the isolation and genetic mapping of YACs at the FLA10 locus on the uni chromosome as well as isolation of a YAC spanning the PF14 locus on chromosome VI . Genetic mapping of YAC end clones by RFLP analyses in interspecific crosses reveals that YACs with a physical size of 150 kb commonly span genetic intervals defined by one or two recombination events in crosses of approximately 20 tetrads . This promises to make chromosomal walking in Chlamydomonas a relatively efficient enterprise . We also describe our development of a method for direct complementation of mutant genes by transformation with amplified wildtype YAC DNA . The use of positional cloning using YACs and this direct functional assay for the presence of a gene in a YAC represent powerful molecular genetic tools enabling the cloning of most any Chlamydomonas gene.

Genomics, 1996 Sep 15, 36(3), 399 - 407
Construction of a YAC contig covering human chromosome 6p22; Malaspina P et al.; A contig covering human chromosome 6p22 that consists of 134 YAC clones aligned based on the presence/absence of 52 DNA markers is presented . This contig overlaps with the 6p23 contig at its telomeric end and with the 6p21.3 contig at its centromeric end . The order of loci within the contig resolves the relative positions of several genetically mapped markers . Among the additional markers used here, there are eight novel PCR assays . The 12 known genes and anonymous ESTs located within the contig establish a first step toward a transcriptional map of this region . The instability of YAC clones observed during this work is also discussed.

Nucleic Acids Res, 1996 Sep 15, 24(18), 3593 - 600
Cloning and characterization of the gene for the somatic form of DNA topoisomerase I from Xenopus laevis; Pandit SD et al.; Two distinct tissue-specific forms of DNA topoisomerase I with M(r) of 165 and 110 kDa have been purified from oocytes and somatic cells respectively of the African frog Xenopus laevis . In this paper, cDNAs encoding a Xenopus topoisomerase I were cloned using PCR primers derived from sequences of yeast and human topoisomerase I . A polypeptide expressed from a portion of the coding sequence was recognized by an antiserum directed against the somatic topoisomerase I that had previously been shown to be unable to cross-react with the oocyte enzyme . Thus, the clone encodes the somatic cell topoisomerase I . An antiserum raised against a synthetic peptide containing the sequence surrounding the active site tyrosine of the somatic topoisomerase I reacts with the enzymes purified from both oocytes and somatic cells, indicating that the two enzymes share some limited sequence homology . RNA blot hybridization showed that oocytes contain an abundant store of somatic topoisomerase I mRNA that is not efficiently polyadenylated in oocytes . This stored RNA contains a consensus cytoplasmic polyadenylation element that is found in a variety of mRNAs that are translationally repressed in oocytes . Microinjection into oocytes of in vitro transcribed mRNA prepared from a Myc-tagged construct of the somatic topoisomerase I sequence is translated to yield a 110 kDa product . This suggests that the oocyte-specific 165 kDa topoisomerase I is not produced by tissue-specific post-translational modification of the somatic topoisomerase I . The oocyte enzyme appears to be produced from a minor mRNA species in oocytes that has not yet been identified.

Nucleic Acids Res, 1996 Sep 15, 24(18), 3576 - 82
TFIIH-mediated nucleotide excision repair and initiation of mRNA transcription in an optimized cell-free DNA repair and RNA transcription assay; Satoh MS et al.; In mammalian cells, mRNA transcription is initiated with the aid of transcription initiation factors . Of these, TFIIH has also been shown to play an essential role in nucleotide excision repair (NER), which is a versatile biochemical pathway that corrects a broad range of DNA damage . Since the dual role of TFIIH is conserved among eukaryotes, including yeast and mammalian cells, the sharing of TFIIH between NER and RNA transcription initiation might provide some survival advantage . However, the functional relationship between NER and RNA transcription initiation through TFIIH is not yet understood . We have developed an optimized cell-free assay which allows us to analyze NER and RNA transcription under identical conditions . In this assay, NER did not compete with RNA transcription, probably because the extracts contained sufficient amounts of TFIIH to support both processes . Thus, NER can be considered functionally independent of RNA transcription initiation despite the fact that both processes use the same factor.

Nucleic Acids Res, 1996 Sep 15, 24(18), 3568 - 75
Interaction of retroviral nucleocapsid proteins with transfer RNAPhe: a lead ribozyme and 1H NMR study; Khan R et al.; In the initiation of reverse transcription in retroviruses, nucleocapsid (NC) protein accelerates the rate of annealing of transfer RNA replication primer to a complementary sequence on the genomic RNA . In this report, we have probed the conformational changes induced by HIV-1 NC protein and domain deletion mutants in a structurally well-characterized transfer RNA, yeast tRNAPhe, as a model for the natural primer . One molar equivalent of recombinant 71 amino acid HIV-1 nucleocapsid protein (NC 1-71) is sufficient to completely inhibit the Pb2(+)-ribozyme activity of tRNAPhe at 25 degrees C, pH 7.0 and 15 mM MgCl2, Zn2 HIV-1 NC proteins which lack one or both flexible terminal domains also inhibit the ribozyme activity . 1H NMR spectra acquired for Mg(2+)-tRNAPhe suggest that NC 1-71 and NC 12-55 (lacking residues 1-11 and 56-71) inhibit the lead-ribozyme activity by only modestly altering the active site region rather than inducing large-scale unfolding of the molecule . In the absence of Mg2+, the extent of destabilization of tRNAPhe is greater but appears to be confined to internal regions of the acceptor and T psi C helices, as evidenced by the selectively enhanced exchange rates for imino protons associated with these base pairs . These findings show that NC destabilizes the folded form of tRNAPhe and by extension, other complex RNAs, in tertiary and secondary structural regions most susceptible to thermally-induced denaturation.

Nucleic Acids Res, 1996 Sep 15, 24(18), 3552 - 9
Gene encoding human Ro-associated autoantigen Y5 RNA; Maraia R et al.; Ro ribonucleoproteins are composed of Y RNAs and the Ro 60 kDa protein . While the Ro 60 kDa protein is implicated in an RNA discard pathway that recognizes 3'-extended 5S rRNAs, the function of Y RNAs remains unknown {O'Brien,C.A . and Wolin,S.L . (1995) Genes Dev . 8,2891-2903} . Y5 RNA occupies a large fraction of Ro 60 kDa protein in human Ro RNPs, contains an atypical 3'-extension not found on other Y RNAs, and constitutes an RNA antigen in certain autoimmune patients {Boulanger et al . (1995) Clin . Exp . Immunol . 99, 29-36} . An overabundance of Y RNA retroposed pseudogenes has previously complicated the isolation of mammalian Y RNA genes . The source gene for Y5 RNA was isolated from human DNA as well as from Galago senegalis DNA . Authenticity of the hY5 RNA gene was demonstrated in vivo and its activity was compared with the hY4 RNA gene that also uses a type 3 promoter for RNA polymerase III . The hY5 RNA gene was subsequently found to reside within a few hundred thousand base pairs of other Y RNA genes and the linear order of the four human Y RNA genes on chromosome 7q36 was determined . Phylogenetic comparative analyses of promoter and RNA structure indicate that the Y5 RNA gene has been subjected to positive selection during primate evolution . Consistent with the proposal of O'Brien and Harley {O'Brian,C.A . and Wolin,S.L . (1992) Gene 116, 285-289}, analysis of flanking sequences suggest that the hY5 RNA gene may have originated as a retroposon.

Genes Dev, 1996 Sep 15, 10(18), 2251 - 64
Human HPK1, a novel human hematopoietic progenitor kinase that activates the JNK/SAPK kinase cascade; Hu MC et al.; The c-Jun amino-terminal kinases (JNKs)/stress-activated protein kinases (SAPKs) play a crucial role in stress responses in mammalian cells . The mechanism underlying this pathway in the hematopoietic system is unclear, but it is a key in understanding the molecular basis of blood cell differentiation . We have cloned a novel protein kinase, termed hematopoietic progenitor kinase 1 (HPK1), that is expressed predominantly in hematopoietic cells, including early progenitor cells . HPK1 is related distantly to the p21(Cdc42/Rac1)-activated kinase (PAK) and yeast STE20 implicated in the mitogen-activated protein kinase (MAPK) cascade . Expression of HPK1 activates JNK1 specifically, and it elevates strongly AP-1-mediated transcriptional activity in vivo . HPK1 binds and phosphorylates MEKK1 directly, whereas JNK1 activation by HPK1 is inhibited by a dominant-negative MEKK1 or MKK4/SEK mutant . Interestingly, unlike PAK65, HPK1 does not contain the small GTPase Rac1/Cdc42-binding domain and does not bind to either Rac1 or Cdc42, suggesting that HPK1 . activation is Rac1/Cdc42-independent . These results indicate that HPK1 is a novel functional activator of the JNK/SAPK signaling pathway.

Blood, 1996 Sep 15, 88(6), 2288 - 97
Inhibition of tartrate-resistant acid phosphatase gene expression by hemin and protoporphyrin IX . Identification of a hemin-responsive inhibitor of transcription; Reddy SV et al.; Tartrate-resistant acid phosphatase (TRAP) is an iron-containing protein encoded by the same gene that codes for uteroferrin, a placental iron transport protein . In human peripheral mononuclear cells, TRAP expression is inhibited by both hemin (ferric protoporphyrin IX) and protoporphyrin IX . Nuclear run-on assays confirmed that this inhibition occurs at the level of gene transcription . Previous studies with mTRAP deletion mutants showed that the hemin effect was dependent on repressor activity in the mTRAP 5'-flanking region at -1846 bp to -1240 bp relative to ATG (Reddy et al, J Bone Mineral Res 10:601, 1995) . We now report that gel shift assays showed a DNA binding protein in nuclear extracts of hemin-treated cells termed hemin response element binding protein (HREBP) . Additional studies have localized the HREBP binding region in the mTRAP 5'-flanking DNA to a 27-bp sequence at -1815 to -1789 bp relative to ATG . A tandem repeat sequence, GAGGC;GAGGC, contained within this DNA segment, was shown to be involved in binding of HREBP . Highly homologous sequences are present in the 5'-flanking region of the hTRAP gene . Binding of HREBP to the mTRAP DNA sequence was inhibited by anti-HAP1 antibodies, indicating homology between the hemin-responsive factor and the yeast heme-dependent transcription factor, HAP1 . A 607-bp segment of the mTRAP 5'-flanking region containing the candidate hemin response element and surrounding sequences conferred hemin regulation on the viral SV40 promoter . Southwestern blotting experiments probing nuclear extracts of hemin-treated U937 cells with the 27-bp binding sequence showed two protein bands at 37 and 133 kD representing candidate HREBPs . A GENINFO search showed several other mammalian genes with tandem GAGGC motifs in noncoding regions, providing the possibility that additional genes may also be regulated by hemin at the level of transcription . These studies provide the first description of a novel iron/hemin-responsive transcriptional regulatory mechanism in mammalian cells.

Blood, 1996 Sep 15, 88(6), 1930 - 5
Cytogenetic and molecular delineation of a region of chromosome 7 commonly deleted in malignant myeloid diseases; Le Beau MM et al.; Loss of a whole chromosome 7 or a deletion of the long arm, del(7q), are recurring abnormalities in malignant myeloid diseases . To determine the location of genes on 7q that are likely to play a role in leukemogenesis, we examined the deleted chromosome 7 homologs in a series of 81 patients with therapy-related or de novo myelodysplastic syndrome or acute myeloid leukemia . Our analysis showed that the deletions were interstitial and that there were two distinct deleted segments of 7q . The majority of patients (65 of 81 {80%}) had proximal breakpoints in bands q11-22 and distal breakpoints in q31-36; the smallest overlapping deleted segment was within q22 . The remaining 16 patients had deletions involving the distal q arm with a commonly deleted segment of q32-33 . To define the proximal deleted segment at 7q22 at a molecular level, we used fluorescence in situ hybridization with a panel of mapped yeast artificial chromosome (YAC) clones from 7q to examine 15 patients with deletion breakpoints in 7q22 . We determined that the smallest overlapping deleted segment is contained in a well-defined YAC contig that spans 2 to 3 Mb . These studies delineate the region of 7q that must be searched to isolate a putative myeloid leukemia suppressor gene, and provide the necessary cloned DNA for more detailed physical mapping and gene isolation.

Virology, 1996 Sep 15, 223(2), 381 - 6
Interaction between the cytoplasmic domains of HIV-1 Vpu and CD4: role of Vpu residues involved in CD4 interaction and in vitro CD4 degradation; Margottin F et al.; The Vpu and CD4 cytoplasmic domains were found, by using a two-hybrid assay in yeast, to interact in the absence of their membrane anchor domains . Studies on several deletion and point mutants revealed that the overall structure of the Vpu cytoplasmic domain is required for this interaction . The Vpu amino acid residues involved in the interaction with CD4 were identified . Deletion of the C-terminal residues of Vpu, required for CD4 degradation, as well as the double mutation on the casein kinase II phosphorylation sites S52N-S56N, also involved in CD4 degradation, resulted in the loss of interaction with CD4 and in the inability to induce CD4 degradation . These results suggest that the ability of Vpu to mediate the degradation of CD4 is linked to its capacity to physically interact with CD4 . However, additional mutagenesis on the S52 site revealed that the interaction between the cytoplasmic domains of Vpu and CD4 is not sufficient for in vitro Vpu-mediated CD4 degradation.

J Biol Chem, 1996 Sep 13, 271(37), 22506 - 13
Interaction of a GRB-IR splice variant (a human GRB10 homolog) with the insulin and insulin-like growth factor I receptors . Evidence for a role in mitogenic signaling; O'Neill TJ et al.; We have utilized the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor . We identified a human cDNA that is a splice variant of the human GRB10 homolog GRB-IR, which we term GRB10/IR-SV1 (for GRB10/GRB-IR splice variant 1) . The protein encoded by the GRB10/IR-SV1 cDNA contains an SH2 domain and a pleckstrin homology domain . Cloning of a full-length human cDNA revealed a predicted coding sequence that was similar to the mouse GRB10 protein, although GRB10/IR-SV1 contained an 80-amino acid deletion . The GRB10/IR-SV1 cDNA is a splice variant of the GRB-IR cDNA such that GRB10/IR-SV1 contains an intact pleckstrin homology domain and a distinct amino terminus . The interaction of GRB10/IR-SV1 with the insulin receptor and the insulin-like growth factor I (IGF-I) receptor is mediated by the SH2 domain, and we show that glutathione S-transferase-SH2 domain fusion proteins interact specifically in vitro with the insulin receptor derived from mammalian cells . The GRB10/IR-SV1 SH2 domain also interacted with an approximately 135-kDa phosphoprotein from unstimulated cell lysates, an interaction that decreased after insulin stimulation . We present evidence that the GRB10/IR-SV1 protein plays a functional role in insulin and IGF-I signaling by showing that microinjection of an SH2 domain fusion protein inhibited insulin- and IGF-I-stimulated mitogenesis in fibroblasts, yet had no effect on mitogenesis induced by epidermal growth factor . Our findings suggest that GRB10/IR-SV1 may serve to positively link the insulin and IGF-I receptors to an uncharacterized mitogenic signaling pathway.

FEBS Lett, 1996 Sep 9, 393(1), 124 - 30
Distribution of bending propensity in DNA sequences; Gabrielian A et al.; Local bending propensity and curvature of DNA can be characterized using a vector description of DNA bendability, based on a set of parameters derived from deoxyribonuclease I (DNase I) cleavage experiments . Two characteristics-arithmetic and vector averages of bendability-were successfully used to predict experimentally known bendable, rigid and curved segments in DNA . A characteristic distribution of bendability is conserved in evolutionarily related kinetoplast sequences . An analysis of the M . genitalium and H . influenzae genomes as well as fragments of human and yeast genomes shows, on the other hand, that highly curved segments--similar to artificially designed curved oligonucleotides--are extremely rare in natural DNA.

Cell, 1996 Sep 6, 86(5), 811 - 22
Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease; Sijbers AM et al.; Nucleotide excision repair, which is defective in xeroderma pigmentosum (XP), involves incision of a DNA strand on each side of a lesion . We isolated a human gene homologous to yeast Rad1 and found that it corrects the repair defects of XP group F as well as rodent groups 4 and 11 . Causative mutations and strongly reduced levels of encoded protein were identified in XP-F patients . The XPF protein was purified from mammalian cells in a tight complex with ERCC1 . This complex is a structure-specific endonuclease responsible for the 5' incision during repair . These results demonstrate that the XPF, ERCC4, and ERCC11 genes are equivalent, complete the isolation of the XP genes that form the core nucleotide excision repair system, and solve the catalytic function of the XPF-containing complex.

Cell, 1996 Sep 6, 86(5), 799 - 810
Inactivation of the HR6B ubiquitin-conjugating DNA repair enzyme in mice causes male sterility associated with chromatin modification; Roest HP et al.; The ubiquitin-conjugating yeast enzyme RAD6 and its human homologs hHR6A and hHR6B are implicated in postreplication repair and damage-induced mutagenesis . The yeast protein is also required for sporulation and may modulate chromatin structure via histone ubiquitination . We report the phenotype of the first animal mutant in the ubiquitin pathway: inactivation of the hHR6B-homologous gene in mice causes male infertility . Derailment of spermatogenesis becomes overt during the postmeiotic condensation of chromatin in spermatids . These findings provide a parallel between yeast sporulation and mammalian spermatogenesis and strongly implicate hHR6-dependent ubiquitination in chromatin remodeling . Since heterozygous male mice and even knockout female mice are completely normal and fertile and thus able to transmit the defect, similar hHR6B mutations may cause male infertility in man.

J Biol Chem, 1996 Sep 6, 271(36), 22030 - 4
Physical interaction of mammalian CDC37 with CDK4; Dai K et al.; CDC37 was originally identified as a Start gene in budding yeast and has been shown to be required for association of CDC28 with cyclins . The exact functional mechanism by which CDC37 promotes this association, however, remains unknown . CDK4 is a cyclin D-dependent kinase that controls progression through G1 of the mammalian cell cycle . We have detected a specific association of CDK4 with the molecular chaperon HSP90 and a 44-kDa protein that we identify as mammalian CDC37 . A physical interaction between CDC37 and CDK4 suggests that CDC37 may regulate the mammalian cell cycle through a direct effect on CDK4 . Association of CDK4 with both CDC37 and HSP90 may also imply a mechanistic link between the functions of CDC37 and HSP90.

J Biol Chem, 1996 Sep 6, 271(36), 21687 - 90
Characterization of the interaction of FKBP12 with the transforming growth factor-beta type I receptor in vivo; Okadome T et al.; The type I transforming growth factor-beta receptor (TbetaR-I) is the efferent component of the receptor complex, which presumably phosphorylates intracellular targets . FKBP12, a binding protein for FK506 and rapamycin, is shown to associate with the cytoplasmic region of TbetaR-I in vitro . In this report, we investigated the interaction of FKBP12 with TbetaR-I in vivo . FKBP12 interacts with TbetaR-I in mammalian cells as well as in yeast . Ligand addition does not affect the interaction, and both constitutively active and kinase-negative mutants of TbetaR-I bind FKBP12 . FKBP12 dissociates from TbetaR-I in the presence of a high concentration of FK506 . The juxtamembrane region of TbetaR-I, containing the major phosphorylation sites by the type II receptor, is required for the interaction . One of the deletion mutants in this region, which was shown to mediate transcriptional response, does not bind FKBP12, suggesting that FKBP12 is not directly involved in TGF-beta signaling . Furthermore TbetaR-I does not phosphorylate FKBP12 in vitro . FKBP12 may not be a direct substrate of TbetaR-I but possibly modulates the TbetaR-I function through its interaction with the regulatory domain of the kinase.

Oncogene, 1996 Sep 5, 13(5), 1043 - 51
Flexibility in the second half-site sequence recognised by the c-Myb R2 domain--in vitro and in vivo analysis; Ording E et al.; The oncoprotein c-Myb is a transcription factor that recognises its specific target sequences through two subdomains . The R3-domain binds the first half-site, YAAC, and plays a dominant role in sequence recognition, while the homologous R2-domain interacts with a more loosely defined sequence in the second half-site . The difficulty in precisely defining a preferred second half-site sequence might reflect the flexible nature of R2 which only attains its fully folded structure upon binding to DNA, a process that might allow the protein to adapt to different half-site sequences . Here we report that shifting the most conserved base in the second half-site, the G6, into position 5 resulted only in a minor reduction of complex stability in vitro . From an analysis of a series of second half-site variants by EMSA and DMS-interference, we conclude that the preferred recognition sequence should be revised to read {YAACNG or YAACGN} . Modeling the structure of c-Myb R2R3 in complex with a GT half-site variant revealed specific interactions with G5 . When second half-site variants were tested in vivo using a sensitive yeast effector-reporter system, both the TG and GT half-site variants were functional mediating c-Myb-dependent transactivation . Unexpectedly, we observed large differences between the best second half-site variants at low levels of c-Myb-effector, the GG variant being five- to fifteen-fold more active in vivo than the single-G half-sites, the GH or HG variants.

Oncogene, 1996 Sep 5, 13(5), 963 - 70
Rapamycin resistance in ataxia-telangiectasia; Beamish H et al.; The gene mutated in the human genetic disorder ataxia-telangiectasia (A-T) has been described recently (Savitsky et al., 1995a) and the complete coding sequence of this gene, ATM, has been reported (Savitsky et al., 1995b) . The derived amino acid sequence demonstrates significant homologies to several proteins containing a phosphatidylinositol 3-kinase (PI3-kinase) domain, including the yeast TOR proteins and the human protein FRAP . Since the TOR and FRAP proteins are targets for the immunosuppressive drug rapamycin, we have investigated the effects of this compound on A-T cells . We report here that 3 A-T cell lines are more resistant than control cells to rapamycin's growth inhibiting effects but were more sensitive to the PI3-kinase inhibitor wortmannin . As expected rapamycin (1 nM) inhibited the rate of exit of control cells from G1 phase but failed to perturb the progression of A-T cells . This difference in cell cycle progress after rapamycin treatment is reflected in ribosomal S6 protein kinase (p70S6k) by both a downward mobility shift on SDS-PAGE and inhibition of activity . Furthermore, the G1 phase cyclin-dependent kinase, cyclin E-cdk2, was rapidly inhibited in control cells post-treatment, whereas in A-T cells it took considerably longer to observe inhibition . There was no evidence that a GST-FKBP12 fusion protein specifically precipitated the ATM protein in the presence of rapamycin in either cell type . These results demonstrate that the ATM protein is not a direct target for rapamycin but its functional loss renders cells more resistant to this compound.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9437 - 42
TRAF5, a novel tumor necrosis factor receptor-associated factor family protein, mediates CD40 signaling; Ishida TK et al.; Signals emanating from CD40 play crucial roles in B-cell function . To identify molecules that transduce CD40 signalings, we have used the yeast two-hybrid system to done cDNAs encoding proteins that bind the cytoplasmic tail of CD40 . A cDNA encoding a putative signal transducer protein, designated TRAF5, has been molecularly cloned . TRAF5 has a tumor necrosis factor receptor-associated factor (TRAF) domain in its carboxyl terminus and is most homologous to TRAF3, also known as CRAF1, CD40bp, or LAP-1, a previously identified CD40-associated factor . The amino terminus has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other members of the TRAF family protein except for TRAF1 . In vitro binding assays revealed that TRAF5 associates with the cytoplasmic tail of CD40, but not with the cytoplasmic tail of tumor receptor factor receptor type 2, which associates with TRAF2 . Based on analysis of the association between TRAF5 and various CD40 mutants, residues 230-269 of CD40 are required for the association with TRAF5 . In contrast to TRAF3, overexpression of TRAF5 activates transcription factor nuclear factor kappa B . Furthermore, amino-terminally truncated forms of TRAF5 suppress the CD40-mediated induction of CD23 expression, as is the case with TRAF3 . These results suggest that TRAF5 and TRAF3 could be involved in both common and distinct signaling pathways emanating from CD40.

Pediatr Pathol Lab Med, 1996 Sep-Oct, 16(5), 713 - 9
The cytology of cerebrospinal fluid associated with neonatal intraventricular hemorrhage; Craver RD; We describe the morphologic features seen in cytocentrifuge preparations of cerebrospinal fluid (CSF) from neonates with intraventricular hemorrhage (IVH) . These CSF specimens from lumbar punctures or ventricular taps often contain degenerated red blood cells with blebs and buds, forming microspherocytes resembling budding yeast or cocci . Hemosiderin-laden macrophages and foamy histiocytes occur in early specimens and persist through multiple specimens . Hematoidin, foreign body giant cells, and CSF eosinophilia are later findings . Brain tissue fragments are frequently seen at the time of ventricular shunt placement . These cytocentrifuge specimens are essentially cytology specimens and in children should be reviewed by a qualified pathologist to interpret the findings in a clinically relevant manner.

Fungal Genet Biol, 1996 Sep, 20(3), 193 - 203
The chsB gene of Aspergillus nidulans is necessary for normal hyphal growth and development; Borgia PT et al.; The chsB gene from Aspergillus nidulans encodes a class III chitin synthase, an enzyme class found in filamentous fungi but not in yeast-like organisms . Using a novel method, we isolated haploid segregants carrying a disrupted chsB allele from heterozygous diploid disruptants . The haploid disruptants grow as minute colonies that do not conidiate . Hyphae from the disruptants have enlarged tips, a high degree of branching, and disorganized lateral walls . The mycelium is not deficient in chitin content and shows no evidence of lysis . The disruptant phenotype is not remedied by osmotic stabilizers . The results indicate that chitin synthesized by the chsB-encoded enzyme does not substantially contribute to the rigidity of the cell wall but is necessary for normal hyphal growth and organization . The properties of the A . nidulans disruptant are similar to those for Neurospora crassa strains with a disrupted chs-1 gene, which also encodes a class III chitin synthase . The morphology of an A . nidulans heterokaryon containing both the wild-type and the disrupted chsB alleles indicates that chsB acts in local areas of the mycelium . The heterokaryon produces conidia of both parental genotypes in nearly equal numbers, indicating that the wild-type chsB gene is not necessary for conidium formation . In addition, we identified and sequenced a second, previously undescribed, homolog of chsB from the closely related opportunistic pathogen, A . fumigatus . The finding of two class III chitin synthase genes in A . fumigatus and a single gene of this class in A . nidulans illustrates limitations of using A . nidulans as a genetic model for A . fumigatus.

Rev Prat, 1996 Sep 1, 46(13), 1617 - 22
{Superficial mucocutaneous mycosis}; Schmutz JL et al.; Superficial mycosis are very common . The frequency in France is 10 to 15% of the population . The reason for the high incidence of this disease is the change of the moral values, rise in promiscuity and increase sports activities . The agents responsible are divided in four groups . Dermatophytes involve keratin of the skin, hairs and nails but never the mucous membranes . They can cause glabrous skin lesion, tinea corporis, tinea capitis or onychomycosis . Yeast are divided in two groups: the candidiasis and Malassezia furfur, the etiologic agent of pityriasis versicolor . Oral mucosa candidiasis is very common in HIV infected patients.

Br J Dermatol, 1996 Sep, 135(3), 475 - 7
Phaeohyphomycosis caused by Exophiala dermatitidis following intra-articular steroid injection; Woollons A et al.; A patient with long-standing rheumatoid arthritis presented with a painful pigmented chronic nodule on the dorsum of the right hand, at the site of intra-articular steroid injections undertaken 5 years previously . Histology showed pigmented fungal elements consistent with phaeohyphomycosis . Cultures yielded black yeast-like colonies, identified as Exophiala dermatitidis and sensitive to itraconazole and amphotericin . A 1-month course of itraconazole resulted in marked clinical improvement but surgical excision and skin grafting were required for complete resolution . Phaeohyphomycosis has been related to inoculation injury but association with intra-articular steroid injection appears hitherto to be unreported.

Ann Hum Genet, 1996 Sep, 60 ( Pt 5), 385 - 9
Human sequences homologous to the gene for the cochlear protein Ocp-II do not map to currently known non-syndromic hearing loss loci; Brown KA et al.; The abundant and almost exclusive expression of OCP-II protein in the mammalian cochlea has fuelled speculation that mutations in the OCP2 gene may result in inherited forms of hearing impairment . We have identified several human sequences related to OCP2 and sublocalised three of these OCP2 related loci to 4q12-p14 or 4p16.2-pter, 5q15-q21.3 and 7p22-q22 by PCR . 2 YACs with sequence consistent with the chromosome 7 locus were also used for FISH analysis and hybridised to chromosome 7q11 . Our data suggest that the cytogenetic localisations of these OCP2 related sequences do not correlate with the precise chromosomal positions of deafness loci so far identified.

J Lipid Mediat Cell Signal, 1996 Sep, 14(1-3), 209 - 14
Effects of acid phospholipids on ARF activities: potential roles in membrane traffic; Kahn RA et al.; ADP-ribosylation factors are a family of approximately 21 kDa GTP binding proteins which have been implicated as ubiquitous regulators of multiple steps in both exocytic and endocytic membrane traffic in mammals and yeast . Reversible membrane associations are thought to be an essential component in the physiological actions of ARF and are regulated by GTP binding . ARFs are unique among the superfamily of GTP binding proteins in having a strict dependence on phospholipids for nucleotide exchange . In addition, ARF proteins were found to bind phospatidylinositol 4,5-bisphosphate (PIP2) specifically . PIP2 was found to increase the rate of GDP dissociation and stabilize the nucleotide-free form of the protein . The previously described requirements for PIP2 in the ARF stimulated phospholipase D (PLD) activity and ARF GTPase activating protein (ARF GAP) assays provide the basis for a model in which PIP2 acts as a cofactor in one or more ARF pathways . There are potentially two distinct phospholipid binding sites each of which are coupled to the nucleotide binding site of ARFs.

Prenat Diagn, 1996 Sep, 16(9), 829 - 35
Prenatal diagnosis of Sanfilippo A syndrome: experience in 35 pregnancies at risk and the use of a new fluorogenic substrate for the heparin sulphamidase assay; Kleijer WJ et al.; We have investigated the use of a 4-methylumbelliferone (MU)-derived artificial substrate, MU-alpha-D-N-sulphoglucosaminide, for the sulphamidase assay in chorionic villi and amniotic fluid cells . In the new two-step enzyme assay, fluorescent MU is released by the successive action of endogenous sulphamidase and an added yeast enzyme preparation which hydrolyses the MU-alpha-glucosaminide intermediate . Optimal conditions for a sensitive, accurate, and convenient procedure for use in the prenatal diagnosis of Sanfilippo A syndrome are described . Previously, prenatal diagnosis of Sanfilippo A syndrome has been achieved by a radioactive sulphamidase assay in chorionic villi or in cultured amniocytes and by two-dimensional electrophoresis of glycosaminoglycans in amniotic fluid . Our experience using these methods in 35 pregnancies at risk is reported . The feasibility of the new fluorogenic assay was evaluated by retrospective testing of stored homogenates of chorionic villi and amniotic fluid cells from 22 pregnancies at risk . Unequivocal assignment of the fetal status in five affected pregnancies and 17 pregnancies with a normal outcome confirms the reliability of the new sulphamidase assay, which is in every respect more convenient than the conventional method using 35S-radiolabelled heparin.

Cell Mol Biol (Noisy-le-grand), 1996 Sep, 42(6), 881 - 8
Phagocytosis occurs in Acanthamoeba castellanii after electroporation; Dybowska U et al.; Acanthamoeba cells treated with an electric discharge were porated and their cytoplasm became accessible to exogenous molecules . Over a broad range of electric field densities low molecular weight markers (trypan blue, ruthenium red), normally unable to penetrate a plasma membrane, gained access to cytoplasm of 80-90% of the cells . Macromolecules (albumin-FITC and IgG-FITC) penetrated into 63-86% of the cells when electroporation was carried out over the range of 1500V/25 microF-400V/250 microF . Pulse labeling with fluorescent markers evidenced that even 3 hrs . after an electric pulse the plasma membrane was still permeable to exogenous fluorescent probes . Following this stage, the pores were gradually closed . The cells electroporated at 400 V/250 microF were able to ingest yeast particles . The uptake of the particles seems to be an active process since it was inhibited by azide and phalloidin . Therefore, the electroporation of Acanthamoeba makes possible the introduction of macromolecules into the cells and subsequent analysis of their effect on active motile processes such as phagocytosis . This should greatly facilitate characterization of the mechanisms by which such processes do occur.

Genome Res, 1996 Sep, 6(9), 862 - 9
Fine mapping of the autosomal dominant juvenile open angle glaucoma (GLC1A) region and evaluation of candidate genes; Sunden SL et al.; Juvenile Open Angle Glaucoma (GLC1A) is an autosomal optic neuropathy that has been localized previously to chromosome 1q . Here we report the fine mapping of the disease region using YACs and a high density of polymorphic microsatellite markers . This study utilized two large JOAG pedigrees genotyped at 36 loci from chromosome 1q21-q31 to refine the GLC1A locus to a approximately 3-cM region flanked by YAC-derived microsatellite markers D1S3665 and D1S3664 . The candidate genes LAMC1, NPR1, and CNR2 were excluded from the region by linkage . Four other genes, SELE, SELL, TXGP1, and APT1LG1, were determined to lie within the critical region through YAC content and linkage mapping . The YAC-STS content map of the critical region provides the groundwork for the construction of a transcription map and the identification of the disease-causing gene.

Genes Chromosomes Cancer, 1996 Sep, 17(1), 1 - 6
Translocation breakpoints upstream of the HMGIC gene in uterine leiomyomata suggest dysregulation of this gene by a mechanism different from that in lipomas; Schoenberg Fejzo M et al.; Uterine leiomyomata are the most common pelvic tumors in women and are the indication for more than 200,000 hysterectomies annually in the United States . Rearrangement of chromosome 12 in bands q14-q15 is characteristic of uterine leiomyomata and other benign mesenchymal tumors, and we identified a yeast artificial chromosome (YAC) spanning chromosome 12 translocation breakpoints in a uterine leiomyoma, a pulmonary chondroid hamartoma, and a lipoma . Recently, we demonstrated that HMGIC, which is an architectural factor mapping within the YAC, is disrupted in lipomas, resulting in novel fusion transcripts . Here, we report on the localization of translocation breakpoints in seven uterine leiomyomata from 10 to > 100 kb upstream of HMGIC by use of fluorescence in situ hybridization . Our findings suggest a different pathobiologic mechanism in uterine leiomyomata from that in lipomas . HMGIC is the first gene identified in chromosomal rearrangements in uterine leiomyomata and has important implications for an understanding of benign mesenchymal proliferation and differentiation.

Biochem Mol Biol Int, 1996 Sep, 40(1), 181 - 8
tRNA(Trp) as cofactor of gelonin, a ribosome-inactivating protein with RNA-N-glycosidase activity . Features required for the cofactor activity; Brigotti M et al.; Purified beef and rabbit liver tRNA(Trp), but not yeast tRNA(Trp), increase the in vitro inactivation of eukaryotic ribosomes by gelonin, a ribosome-inactivating protein with RNA-N-glycosidase activity on 28S rRNA . Aminoacylation and stepwise trimming of the 3'-end of bovine tRNA(Trp) affect the cofactor activity, the most active species being that shortened by the last two nucleotides . ATP only moderately increases the activity of purified mammalian tRNA(Trp) and in this increase the cognate aminoacyl-tRNA synthetase apparently has no role . Mobility shift experiments indicate that bovine tRNA(Trp) binds both to ribosomes and to gelonin and favours the dissociation of gelonin from ribosomes.

J Med Genet, 1996 Sep, 33(9), 795 - 7
A 3 1/2 year old girl with distal trisomy 19q defined by FISH; James C et al.; A 3 1/2 year old girl was evaluated because of developmental delay . Short stature was evident with height between the 3rd and 10th centiles, while weight and head circumference were on the 50th centile . Dysmorphic features consisted of a high bossed forehead, pointed short ear lobes, small nose, bilateral convergent strabismus, left simian crease, a gap between the first and second toes bilaterally, mild clinodactyly, and a broad, barrel shaped thorax . Cytogenetic investigations showed an unbalanced karyotype, 46,XX,10q+, which was de novo in origin . Fluorescence in situ hybridisation (FISH) using three library probes (from chromosomes 10, 19, and 19q) and a YAC probe (from 10q telomere) showed that the additional material on 10q was derived from chromosome 19q . The patient had an unbalanced translocation, 46,XX,-10,+der(10)t(10;19)(q26.3; q13.3), which resulted in distal trisomy 19q . Few other cases of proven distal trisomy 19q are available for comparison of clinical features.

Genetics, 1996 Sep, 144(1), 369 - 82
Dynamics of plant mitochondrial genome: model of a three-level selection process; Albert B et al.; The plant mitochondrial genome is composed of a set of molecules of various sizes that generate each other through recombination between repeated sequences . Molecular observations indicate that these different molecules are present in an equilibrium state . Different compositions of molecules have been observed within species . Recombination could produce deleted molecules with a high replication rate but bearing little useful information for the cell (such as "petite" mutants in yeast) . In this paper we use a multilevel model to examine selection among rapidly replicating incomplete molecules and relatively slowly replicating complete molecules . Our model simulates the evolution of mitochondrial information through a three-level selection process including intermolecular, intermitochondrial, and intercellular selection . The model demonstrates that maintenance of the mitochondrial genome can result from multilevel selection, but maintenance is difficult to explain without the existence of selection at the intermitochondrial level . This study shows that compartmentation into mitochondria is useful for maintenance of the mitochondrial information . Our examination of evolutionary equilibria shows that different equilibria (with different combinations of molecules) can be obtained when recombination rates are lower than a threshold value . This may be interpreted as a drift-mutation balance.

Hum Mol Genet, 1996 Sep, 5(9), 1311 - 8
Spinocerebellar ataxia type-1 and spinobulbar muscular atrophy gene products interact with glyceraldehyde-3-phosphate dehydrogenase; Koshy B et al.; Spinocerebellar ataxia type1 (SCA1) is one of several neurodegenerative disorders caused by expansions of translated CAG trinucleotide repeats which code for polyglutamine in the respective proteins . Most hypotheses about the molecular defect in these disorders suggest a gain of function, which may involve interactions with other proteins via the expanded polyglutamine tract . In this study we used ataxin-1, the SCA1 gene product, as a bait in the yeast two-hybrid system and identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase as an ataxin-1 interacting protein . In addition, the yeast two hybrid data demonstrate that wild type and mutant ataxin-1 form homo and heterodimers . Physical interaction between GAPDH and ataxin-1 was also demonstrated in vitro . To investigate if GAPDH might interact with other glutamine repeat-containing proteins involved in neurodegenerative disorders, we tested its binding to the androgen receptor which is mutated in spinobulbar muscular atrophy . The androgen receptor interacts with GAPDH both in the yeast two-hybrid system and in vitro . The binding of both ataxin-1 and the androgen receptor to GAPDH does not vary with the length of the polyglutamine tract . While provocative, these findings do not address the selective neuronal loss in each of these disorders in light of the wide expression patterns of GAPDH and the respective polyglutamine containing proteins . Nonetheless, such interactions may increase the susceptibility of specific neurons to a variety of insults and initiate degeneration.

Brain Res Mol Brain Res, 1996 Sep 1, 40(2), 195 - 202
Use of a two-hybrid system to investigate molecular interactions of GAP-43; Chao S et al.; We used the 'interaction trap' (two-hybrid system) to identify polypeptides that interact with the neuronal phosphoprotein, GAP-43, in an intracellular environment . GAP-43 (neuromodulin, B-50, F1), a protein kinase C (PKC) substrate important for the growth and plasticity of neuronal connections, has been implicated in vitro in several signal transduction pathways . In the yeast-based cloning system, the only strong interaction that was detected between GAP-43 and the calcium effector protein, calmodulin (CaM) . PKC phosphorylates GAP-43 on serine 41 . When we changed this serine to an aspartate residue to mimic constitutive phosphorylation, the interaction with CaM was blocked . Surprisingly, the N-terminal third of GAP-43 alone bound CaM more strongly than did intact GAP-43, suggesting that the protein's C-terminus may play a role in modulating the interaction with CaM . These results, along with other recent findings, suggest a novel role for the interaction between GAP-43 and CaM.

Trends Biochem Sci, 1996 Sep, 21(9), 346 - 50
The multiple roles of transcription/repair factor TFIIH; Svejstrup JQ et al.; TFIIH is by far the most complex of the basal RNA polymerase II transcription factors . It is a protein kinase, a bi-directional DNA helicase and is essential for both transcription and nucleotide excision repair (NER) . Furthermore, the factor can activate cyclin-dependent kinases and so might play a role in cell-cycle regulation . The recent elucidation of the subunit composition of TFIIH has shown an extraordinary conservation of its structure from yeast to human.

Trends Biochem Sci, 1996 Sep, 21(9), 327 - 35
The role of general initiation factors in transcription by RNA polymerase II; Roeder RG; Transcription initiation on protein-encoding genes represents a major control point for gene expression in eukaryotes, and is mediated by RNA polymerase II and a surprisingly complex array of general initiation factors (TFIIA, -B, -D, -E, -F and -H) that are highly conserved from yeast to man . Elucidation of structural and functional features of these factors on model promoters has revealed insights into biochemical mechanisms and provides a basis for understanding their regulation on diverse promoters by gene- and cell-specific activators.

Plant Cell, 1996 Sep, 8(9), 1465 - 76
Distinct classes of cdc2-related genes are differentially expressed during the cell division cycle in plants; Fobert PR et al.; cdc2 and several related genes encode the catalytic subunits of cyclin-dependent kinases, which have been implicated in a number of cellular processes, including control of cell division . As a first step in exploring their function in plants, we isolated four cdc2-related genes from Antirrhinum . Two genes, cdc2a and cdc2b, encode proteins that contain a perfectly conserved PSTAIRE motif characteristic of cdc2 homologs, whereas the products of the two remaining genes, cdc2c and cdc2d, appear to represent a new subclass of proteins that have so far only been identified in plants . Transcripts of these novel genes were localized in isolated cells dispersed throughout actively dividing regions of the inflorescence . This localization is consistent with accumulation that is specific to particular phases of the cell cycle . Correlating cell labeling with nuclear condensation and double-labeling experiments using cdc2 and histone H4 as probes indicated that cdc2c transcripts accumulate during S phase as well as during the G2 and M transition, whereas cdc2d expression was specific to the G2 and M phases . All cells labeled with cdc2d also contained cdc2c label, Indicating that expression of cdc2d completely overlapped with that of cdc2c . Transcripts of cdc2a and cdc2b were detected in all cells within actively dividing regions, but at levels that were only slightly higher than those observed in nondividing areas . These transcripts did not appear to accumulate in a cell cycle-specific fashion . The genes cdc2a and cdc2b were able to partially complement a yeast cdc2 mutation, although all four genes appeared to interfere with the sizing mechanism of yeast cells . We propose that plants contain at least two classes of cdc2-related genes that differ in structure, expression, and perhaps function.

Br J Cancer, 1996 Sep, 74(6), 863 - 70
High levels of loss at the 17p telomere suggest the close proximity of a tumour suppressor; White GR et al.; High levels of loss of distal markers on 17p13.3 in breast cancer suggested the presence within the region of at least one tumour-suppressor gene . Here we describe the derivation of two biallelic polymorphisms from the 17p telomeric yeast artificial chromosome (YAC) TYAC98 . Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex PCR analysis demonstrated that the high level of allelic imbalance observed in breast tumours represented loss of constitutional heterozygosity (LOH) and that this LOH extended to the telomere . Lung carcinoma (but not Wilms' tumour)-derived DNA again revealed a high level of loss of subtelomeric 17p sequences . Telomeric microsatellite polymorphisms from other chromosome arms did not show such elevated loss in either tumour type . This suggested that the 17p loss observed did not reflect a general telomeric instability and provided further evidence for the presence of a breast cancer tumour-suppressor gene in the distal region of 17p13.3.

Genomics, 1996 Sep 1, 36(2), 328 - 36
Identification of genes from a 500-kb region at 7q11.23 that is commonly deleted in Williams syndrome patients; Osborne LR et al.; Williams syndrome (WS) is a multisystem developmental disorder caused by the deletion of contiguous genes at 7q11.23 . Hemizygosity of the elastin (ELN) gene can account for the vascular and connective tissue abnormalities observed in WS patients, but the genes that contribute to features such as infantile hypercalcemia, dysmorphic facies, and mental retardation remain to be identified . In addition, the size of the genomic interval commonly deleted in WS patients has not been established . In this study we report the characterization of a 500-kb region that was determined to be deleted in our collection of WS patients . A detailed physical map consisting of cosmid, P1 artificial chromosomes, and yeast artificial chromosomes was constructed and used for gene isolation experiments . Using the techniques of direct cDNA selection and genomic DNA sequencing, three known genes (ELN, LIMK1, and RFC2), a novel gene (WSCR1) with homology to RNA-binding proteins, a gene with homology to restin, and four other putative transcription units were identified . LIMK1 is a protein kinase with two repeats of the LIM/double zinc finger motif, and it is highly expressed in brain . RFC2 is the 40-kDa ATP-binding subunit of replication factor C, which is known to play a role in the elongation of DNA catalyzed by DNA polymerase delta and epsilon . LIMK1 and WSCR1 may be particularly relevant when explaining cognitive defects observed in WS patients.

Genomics, 1996 Sep 1, 36(2), 316 - 9
Isolation of three testis-specific genes (TSA303, TSA806, TSA903) by a differential mRNA display method; Ozaki K et al.; We isolated three human testis-specific genes by a differential mRNA display method . The cDNAs contained open reading frames of 1620, 453, and 333 nucleotides, encoding 540, 151, and 111 amino acids, respectively . The first of these genes, designated TSA303, encodes a novel protein homologous to TCP20, one of the subunits of the human TRiC chaperonin complex that can bind newly synthesized or unstable folding intermediates of polypeptides and assist substrate proteins in folding, assembly, and transport . The second, TSA806, encodes a novel protein containing 3.3 contiguous repeats of the cdc10/swi6 (ankyrin) motif that was originally found in products of cell cycle control genes of yeast and cell fate determination genes in Drosophila and Caenorhabditis elegans . The third gene, TSA903, encodes a protein homologous to the C-terminal region of murine uridine monophosphate kinase . Northern blot analysis confirmed that in 16 human adult tissues examined, each of these genes was expressed specifically in the testis . From the results of cDNA screening of nearly 1 million plaques, the abundance of each transcript in a preparation of total mRNA was estimated as 0.0004% (TSA303), 0.0006% (TSA806), and 0.0002% (TSA903) . Our results imply that the differential display method is a powerful tool for isolation of tissue-specific genes even if they are expressed at a level as low as 1 in several hundred thousand to a million molecules of total mRNA.

Genomics, 1996 Sep 1, 36(2), 271 - 9
UBL1, a human ubiquitin-like protein associating with human RAD51/RAD52 proteins; Shen Z et al.; The RAD51/RAD52-dependent DNA repair pathway is involved in DNA recombination and DNA double-strand break repair in yeast . Although many proteins in the RAD51/RAD52-dependent DNA repair pathway have been identified in yeast, a novel protein(s) that functions with RAD51/RAD52 may also exist in humans . Using a yeast two-hybrid system, we have identified a 12-kDa protein that associates with the human RAD51 and RAD52 proteins . This protein shares significant amino acid homology with the yeast protein SMT3, which functionally associates with the yeast mitosis fidelity protein MIF2 . It also shares moderate homology with ubiquitin and several other proteins, including the N-terminus of the RAD23 protein and a ubiquitin cross reacting protein . Therefore, the gene is tentatively designated UBL1 for ubiquitin-like 1 . The UBL1 mRNA is expressed in many human tissues, most highly in testis . The UBL1 gene is mapped to chromosome 2q32.2-q33, and a related sequence may be located on chromosome 1q23-q25.

Genomics, 1996 Sep 1, 36(2), 240 - 51
Localization of eight additional genes in the human major histocompatibility complex, including the gene encoding the casein kinase II beta subunit (CSNK2B); Albertella MR et al.; A wide range of autoimmune and other diseases are known to be associated with the major histocompatibility complex . Many of these diseases are linked to the genes encoding the polymorphic histocompatibility antigens in the class I and class II regions, but some appear to be more strongly associated with genes in the central 1100-kb class III region, making it important to characterize this region fully for the presence of novel genes . An approximately 220-kb segment of DNA in the class III region separating the Hsp70 (HSPA1L) and BAT1 (D6S8IE) genes, which was previously known to contain 14 genes, has been analyzed for the presence of additional genes . Genomic DNA fragments spanning the gaps between the known genes were used as probes to isolate cDNAs corresponding to five new genes within this region . Evidence from Northern blot analysis and exon trapping experiments that suggested the presence of at least two more new genes was also obtained . Partial cDNA and complete exonic genomic sequencing of one of the new genes has identified it as the casein kinase II beta subunit (CSNK2B) . Two of the other novel genes lie within a region syntenic to that implicated in susceptibility to experimental allergic orchitis in the mouse, an autoimmune disease of the testis, and represent additional candidates for the Orch-1 locus associated with this disease . In addition, characterization of the 13-kb intergenic gap separating the RD (D6545) and G11 (D6S60E) genes has revealed the presence of a gene encoding a 1246-amino-acid polypeptide that shows significant sequence similarity to the yeast anti-viral Ski2p gene product.

Fundam Appl Toxicol, 1996 Sep, 33(1), 100 - 8
Contribution of mast cell mediators to alterations in macrophage function after malathion administration; Rodgers K et al.; Previous studies showed that acute administration of noncholinergic doses of malathion increased macrophage function and the generation of a primary humoral immune response to a T-dependent antigen and caused mast cell degranulation . Recent studies using mast cell-deficient mice showed that the presence of mast cells was necessary for the increase in macrophage function observed after oral administration of malathion, and reconstitution with bone marrow-derived mast cells restored the ability of malathion to increase macrophage function . In the present study, the contribution of mast cell mediators to alterations in macrophage function after oral administration of malathion was examined . Controls in this study included the effect of the agent to be examined on resident peritoneal macrophages and macrophages elicited with pristane, an agent that can stimulate macrophages in the absence of mast cells . Coadministration of intraperitoneal cromolyn, a stabilizer of mast cell membranes, with oral malathion blocked the ability of malathion to increase macrophage function as measured by the generation of respiratory burst activity, the phagocytosis of opsonized yeast, and the production of cathepsin D . On the other hand, administration of cromolyn to mice whose macrophage function was stimulated with pristane did not affect the observed increases in macrophage function . As oral administration of malathion caused histamine release, the ability of a histamine receptor antagonist, pyrilamine, to alter the response of peritoneal macrophages to oral administration of malathion was also examined . Intraperitoneal administration of pyrilamine partially blocked the effects of oral administration of malathion on peritoneal macrophage function, but did not affect the function of resident or pristane-elicited peritoneal macrophages . These data suggest that mediators from mast cells contribute to the elevation in macrophage function observed after oral malathion administration.

Nucleic Acids Res, 1996 Sep 1, 24(17), 3472 - 3
A fluorescence-labeling method for sequencing small RNA on polyacrylamide gel; Wu TP et al.; A practical fluorescence-labeling method for sequencing small RNAs by the traditional 'direct read out' on polyacrylamide gel electrophoresis was established . The 3' terminus of RNA was oxidized into dialdehyde by sodium periodate and then labeled with fluorescein-5-thiosemicarbazide through the condensation reaction between carbazide and aldehyde . The fluorescence-labeled RNA was partially degraded enzymatically and fractionated by polyacrylamide gel electrophoresis . The fluorescent bands were visualised by ultraviolet photography . A partial sequence of yeast 5S rRNA was determined . The result indicates that this method can be used in sequencing small RNAs rapidly, conveniently and safely.

Nucleic Acids Res, 1996 Sep 1, 24(17), 3467 - 8
Directed mutagenesis of YAC-cloned DNA using a rapid, PCR-based screening protocol; Tucker RM et al.; We have developed a system which facilitates the rapid modification of yeast artificial chromosome (YAC) insert DNA . Specific modifications, such as deletions, insertions and point mutations, can be generated by a two-step allele replacement method using the yeast translational suppressor, SUP4-o, as both a positive and negative selection . The introduction of the SUP4-o gene was successful in 4 out of 24 selected transformant colonies, while the subsequent homologous elimination occurred in 2 out of 30 colonies . The use of a simple, short-range PCR assay rapidly identified the correct events among the genetically selected isolates and should be generally applicable to YAC modifications.

Nucleic Acids Res, 1996 Sep 1, 24(17), 3399 - 406
Fe.bleomycin as a probe of RNA conformation; Holmes CE et al.; Two crystallographically defined tRNAs, yeast tRNAAsp and tRNAPhe, were used as substrates for oxidative cleavage by Fe.bleomycin to facilitate definition at high resolution of the structural elements in RNAs conducive to bleomycin binding and cleavage . Yeast tRNAAsp underwent cleavage at G45 and U66; yeast tRNAPhe was cleaved at four sites, namely G19, A31, U52 and A66 . Only two of these six sites involved oxidative cleavage of a 5'-G.Pyr-3' sequence, but three sites were at the junction between single- and double-stranded regions of the RNA, consistent with a binding model in which the bithiazole + C-terminal substituent of bleomycin bind to minor groove structures on the RNA . Also studied were four tRNA transcripts believed on the basis of biochemical and chemical mapping experiments to share structural elements in common with the mature tRNAs . Cleavage of these tRNAs by Fe.bleomycin gave patterns of cleavage very different from each other and than those of the mature tRNAs . This observation suggests strongly that Fe.bleomycin cannot be used for chemical mapping in the same fashion as more classical reagents, such as Pb2+ or dimethyl sulfate . However, the great sensitivity of Fe.bleomycin to changes in nucleic acid structure argues that those species which do show similar patterns of cleavage must be very close in structure.

Nucleic Acids Res, 1996 Sep 1, 24(17), 3370 - 80
Mutational analysis of the human nucleotide excision repair gene ERCC1; Sijbers AM et al.; The human DNA repair protein ERCC1 resides in a complex together with the ERCC4, ERCC11 and XP-F correcting activities, thought to perform the 5' strand incision during nucleotide excision repair (NER) . Its yeast counterpart, RAD1-RAD10, has an additional engagement in a mitotic recombination pathway, probably required for repair of DNA cross-links . Mutational analysis revealed that the poorly conserved N-terminal 91 amino acids of ERCC1 are dispensable for both repair functions, in contrast to a deletion of only four residues from the C-terminus . A database search revealed a strongly conserved motif in this C-terminus sharing sequence homology with many DNA break processing proteins, indicating that this part is primarily required for the presumed structure-specific endonuclease activity of ERCC1 . Most missense mutations in the central region give rise to an unstable protein (complex) . Accordingly, we found that free ERCC1 is very rapidly degraded, suggesting that protein-protein interactions provide stability . Survival experiments show that the removal of cross-links requires less ERCC1 than UV repair . This suggests that the ERCC1-dependent step in cross-link repair occurs outside the context of NER and provides an explanation for the phenotype of the human repair syndrome xeroderma pigmentosum group F.

Arch Biochem Biophys, 1996 Sep 1, 333(1), 260 - 6
cDNA cloning of a chick homologue of human ATPase complex subunit 4, quantitative tissue distribution and tertiary structure comparison of the ATPase domain to RecA; Singh I et al.; The ocular lens consists of a single layer of epithelial cells on its anterior surface and underlying fiber cells, which are derived from the epithelial cells by differentiation and make up the bulk of the lens . Because lens cells are segregated by age and stage of differentiation, we are using this tissue to study the role of the proteasome in differentiation . The purpose of this study is to corroborate the ATPase function of chick subunit 4 (cS4) and assess the levels of the mRNA in the differentiating lens relative to other tissues . We have generated a computer model of the tertiary structure of the ATPase domain of the cS4 of the ATPase complex that regulates the 20S proteasome . The predicted polypeptide from the cloned cDNA of cS4 (440 residues) had a calculated molecular mass of 49,182 and is 98 and 73% identical to human and yeast S4 protein sequences, respectively . A computer search for comparison with known proteins in GenBank showed that the cS4 protein sequence has a conserved region of about 200 amino acid residues including an ATP/GTP binding site and a mitochondrial energy transfer proteins signature sequence . Based on secondary structure, the computer-generated model of the ATPase domain is comparable to that of RecA, with a root mean square deviation of 0.851 from the RecA triad . mRNA in the 14-day-old chick embryo lens is derived primarily (90%) from differentiating cells . The level of cS4 mRNA determined by quantitative RT/PCR in this differentiating tissue was comparable to the cS4 mRNA levels in chick liver, heart, and brain.

Curr Biol, 1996 Sep 1, 6(9), 1104 - 13
Phosphorylated nitrate reductase from spinach leaves is inhibited by 14-3-3 proteins and activated by fusicoccin; Moorhead G et al.; BACKGROUND . Nitrate reductase (NR) in leaves is rapidly inactivated in the dark by a two-step mechanism in which phosphorylation of NR on the serine at position 543 (Ser543) promotes binding to nitrate reductase inhibitor protein (NIP) . The eukaryotic 14-3-3 proteins bind to many mammalian signalling components (Raf-1, Bcr, phosphoinositide 3-kinase, protein kinase C, polyomavirus middle-T antigen and Cdc25), and are implicated in the timing of mitosis, DNA-damage checkpoint control, exocytosis, and activation of the plant plasma-membrane H+-ATPase by fusicoccin . Their dimeric, saddle-shaped structures support the proposal that 14-3-3 proteins are 'adaptors' linking different signalling proteins, but their precise functions are still a mystery . RESULTS . We purified NIP to homogeneity and established by means of amino-acid sequencing that it is a mixture of several 14-3-3 isoforms . Mammalian and yeast 14-3-3 proteins were just as effective as NIP at inhibiting phosphorylated NR . The sequence around Ser543, the phosphorylation site in NR, is strikingly similar to the sequences around the phosphoserine residues (Ser259 and Ser621) of mammalian Raf-1 that interact with 14-3-3 proteins . We found that NIP activity was blocked by a synthetic phosphopeptide corresponding to residues 251-266 of Raf . Fusicoccin also blocked NIP activity, and plant plasma-membrane H+-ATPases were activated by either fusicoccin, the phosphoserine259-Raf-1 peptide, or protein phosphatase 2A . CONCLUSIONS . Our findings establish that the mechanism of inactivation of NR involves the phosphorylation of Ser 543 followed by interaction with one or more plant 14-3-3 proteins . These results support the idea of a common mechanism for binding of 14-3-3 to its targets in all eukaryotes, and suggest that the phosphoserine259-Raf-1 peptide and fusicoccin may be of general use for disrupting the interaction of 14-3-3 with its target proteins . We propose that the plant plasma-membrane H+-ATPase is regulated in an analogous manner to NR-NIP, and speculate that 14-3-3 proteins provide a link between 'sensing' the activity state of NR and signalling to other cellular processes in plants.

Genes Dev, 1996 Sep 1, 10(17), 2179 - 88
Oskar protein interaction with Vasa represents an essential step in polar granule assembly; Breitwieser W et al.; The posterior pole plasm of the Drosophila egg contains the determinants of abdominal and germ-cell fates of the embryo . Pole plasm assembly is induced by oskar RNA localized to the posterior pole of the oocyte . Genetics has revealed three additional genes, staufen, vasa, and tudor, that are also essential for pole plasm formation . Staufen protein is required for both oskar RNA localization and translation . Vasa and Tudor are localized dependent on Oskar protein and are required to accumulate Oskar protein stably at the posterior pole . We have explored interactions between these gene products at the molecular level and find that Oskar interacts directly with Vasa and Staufen, in a yeast two-hybrid assay . These interactions also occur in vitro and are affected by mutations in Oskar that abolish pole plasm formation in vivo . Finally, we show that in the pole plasm, Oskar protein, like Vasa and Tudor, is a component of polar granules, the germ-line-specific RNP structures . These results suggest that the Oskar-Vasa interaction constitutes an initial step in polar granule assembly . In addition, we discuss the possible biological role of the Oskar-Staufen interaction.

J Acquir Immune Defic Syndr Hum Retrovirol, 1996 Sep, 13(1), 18 - 22
Impairment of phagosome-lysosome fusion in HIV-1-infected macrophages; Moorjani H et al.; Phagosome-lysosome fusion is critical for intracellular killing of most organisms and is inhibited by some viruses, notably influenza . We explored the effects of infection in vitro with HIV-1 (IIIB or Ada-M) on phagosome-lysosome fusion in blood monocyte-derived macrophages . After 8 days of infection, fusion was assessed from the fluorescence change occurring up to 2 h after labeling the lysosome compartment with acridine orange and loading of phagosomes with opsonized yeast . Compared with mock-infected control macrophages, the proportion of cells showing fusion after infection was reduced from a mean of 70% to a mean of 47% (p = 0.0001) . Inhibition was seen with heat-killed HIV-1 IIIB but not virus-free filtrate . It was mimicked by recombinant gp 120 and blocked by soluble CD4 or antibody to CD4 but not by a neutralizing antibody to the V3 loop of gp 120 . The inhibitory effect was seen 8 days after the original, transient exposure to gp 120 . These results suggest that a lasting abnormality of phagosome-lysosome fusion results from interaction between gp 120 and CD4, contributing, perhaps, to the increased susceptibility to opportunistic infections of people infected with HIV.

Appl Environ Microbiol, 1996 Sep, 62(9), 3165 - 70
Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata; Saha BC et al.; Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose) . An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography . The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration . It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G) . The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively . The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml) . Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose . The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM) . Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11% . Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%) . Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).

Mol Pharmacol, 1996 Sep, 50(3), 506 - 11
Calcineurin mutants render T lymphocytes resistant to cyclosporin A; Zhu D et al.; The immunosuppressants cyclosporin A (CsA) and FK506 have been widely used to prevent and treat graft rejection after human organ and tissue transplantations . CsA and FK506 associate with intracellular binding proteins (i.e., CsA with cyclophilin A and FK506 with FKBP12) to form protein/drug complexes that suppress the immune system by preventing activation of T cells in response to antigen presentation . The common target of CsA and FK506 is calcineurin, a Ca2+/calmodulin-regulated, serine/threonine-specific protein phosphatase that regulates the nuclear import of a transcription factor, NF-AT, required for expression of T cell activation genes . In previous studies, we identified calcineurin mutations that block binding by the cyclophilin A/CsA or FKBP12/FK506 complexes and thereby render yeast cells resistant to the antifungal effects of CsA or FK506 . In this report, we demonstrate that the corresponding mutations in murine calcineurin render the T cell receptor signal transduction cascade CsA resistant in human Jurkat T cells . Our findings support the recently determined calcineurin X-ray crystal structure, provide evidence that calcineurin is the only CsA-sensitive component limiting signaling from the T cell receptor to the nucleus, and suggest a means to render cells and tissues resistant to the toxic side effects of CsA and FK506.

Nat Genet, 1996 Sep, 14(1), 102 - 5
Mutation of MSH3 in endometrial cancer and evidence for its functional role in heteroduplex repair; Risinger JI et al.; Many human tumours have length alterations in repetitive sequence elements . Although this microsatellite instability has been attributed to mutations in four DNA mismatch repair genes in hereditary nonpolyposis colorectal cancer (HNPCC) kindreds, many sporadic tumours exhibit instability but no detectable mutations in these genes . It is therefore of interest to identify other genes that contribute to this instability . In yeast, mutations in several genes, including RTH and MSH3, cause microsatellite instability . Thus, we screened 16 endometrial carcinomas with microsatellite instability for alterations in FEN1 (the human homolog of RTH) and in MSH3 (refs 12-14) . Although we found no FEN1 mutations, a frameshift mutation in MSH3 was observed in an endometrial carcinoma and in an endometrial carcinoma cell line . Extracts of the cell line were deficient in repair of DNA substrates containing mismatches or extra nucleotides . Introducing chromosome 5, encoding the MSH3 gene, into the mutant cell line increased the stability of some but not all microsatellites . Extracts of these cells repaired certain substrates containing extra nucleotides, but were deficient in repair of those containing mismatches or other extra nucleotides . A subsequent search revealed a second gene mutation in HHUA cells, a missense mutation in the MSH6 gene . Together the data suggest that the MSH3 gene encodes a product that functions in repair of some but not all pre-mutational intermediates, its mutation in tumours can result in genomic instability and, as in yeast, MSH3 and MSH6 are partially redundant for mismatch repair.

Curr Genet, 1996 Sep, 30(4), 338 - 46
Molecular characterization of U3 small nucleolar RNA from the early diverging protist, Euglena gracilis; Greenwood SJ et al.; U3 small nucleolar RNA (snoRNA) has been isolated from Euglena gracilis, an early diverging protist, and its primary sequence determined . Although this 180-nucleotide-long RNA is considerably smaller than its homolog in vertebrate animals, it contains the conserved sequence blocks (boxes A, Ao, B, C and D) characteristic of U3 snoRNAs from other organisms . A secondary structure can be modelled that displays many of the salient features found in published core structures of vertebrate, yeast and trypanosome U3 snoRNAs . The functional significance of this proposed secondary structure is discussed in relation to the role E . gracilis U3 snoRNA may have in pre-rRNA processing in this organism . Multiple expressed species of E . gracilis U3 snoRNA were found to differ in nucleotide sequence at a number of positions; some of these differences alter pairing in the proposed secondary structure . Analysis of E . gracilis genomic DNA revealed a complex pattern of U3-hybridizing sequences that parallels the multiplicity of expressed species of U3 snoRNA revealed by transcript analysis.

Mol Cell Biol, 1996 Sep, 16(9), 4915 - 22
Composite patterns in neutral/neutral two-dimensional gels demonstrate inefficient replication origin usage; Kalejta RF et al.; The neutral/neutral two-dimensional (2-D) gel replicon mapping technique has been used to great advantage to localize and characterize origins of replication . Interestingly, many yeast origins display a composite pattern consisting of both a bubble arc and a single-fork arc . Moreover, in every instance in which neutral/neutral 2-D gels have been used to analyze origins in higher eukaryotic cells, two or more adjacent fragments display these composite patterns . We believe that composite patterns signal inefficient origin usage in yeast cells because the replicators in question are not active in every cell cycle and in higher eukaryotic replicons because initiation sites are chosen from among many potential sites lying within a zone . However, others have suggested that the single-fork arcs in these composite gel patterns arise from nicking activity that converts replication bubbles to branched structures that comigrate with bona fide single forks . Here, we have used three different replicon mapping strategies to show that broken simian virus 40 replication bubbles trace unique arcs that are clearly distinguishable from classic, intact single forks . Thus, it is likely that composite 2-D gel patterns represent origins that are inefficiently utilized.

Mol Cell Biol, 1996 Sep, 16(9), 4639 - 47
TATA-box DNA binding activity and subunit composition for RNA polymerase III transcription factor IIIB from Xenopus laevis; McBryant SJ et al.; The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-associated factors (TAFs) . Previous studies have shown that DNA oligonucleotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exposed in TFIIIB . We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription inhibition assays and a gel mobility shift assay . Gel shift competition assays with mutant and nonspecific DNAs demonstrate the specificity of the TFIIIB-TATA box DNA complex . The apparent dissociation constant for this protein-DNA interaction is approximately 0.4 nM, similar to the affinity of yeast TBP for the same sequence . TFIIIB transcriptional activity and TATA-box binding activity cofractionate during a series of four ion-exchange chromatographic steps, and reconstituted transcription reactions demonstrate that the TATA-box DNA-protein complex contains TFIIIB TAF activity . Polypeptides with apparent molecular masses of 75 and 92 kDa are associated with TBP in this complex . These polypeptides were renatured after elution from sodium dodecyl sulfate-gels and tested individually and in combination for TFIIIB TAF activity . Recombinant TBP along with protein fractions containing the 75- and 92-kDa polypeptides were sufficient to reconstitute TFIIIB transcriptional activity and DNA binding activity, suggesting that Xenopus TFIIIB is composed of TBP along with these polypeptides.

Biopolymers, 1996 Sep, 39(3), 353 - 65
Catalytic subunit of cAMP-dependent protein kinase: electrostatic features and peptide recognition; Tsigelny I et al.; The electrostatic field was calculated for the mammalian cAMP-dependent protein kinase (PKA) catalytic subunit (C-subunit) complexed with a 20-residue peptide from a heat stable protein kinase inhibitor (PKI: 5-24) . The electrostatic field was also calculated for the C-subunit complexed with a modeled heptapeptide substrate that has been used extensively in structure/function studies for the C-subunit . Perturbations in the electrostatic free energy were calculated when single ionizable active site residues were mutated to alanine . These perturbations in electrostatic free energy were correlated to changes in the binding energy measured in a charge-to-alanine scan of the homologous yeast C-subunit by M . J . Zoller and C . S . Gibbs {(1991) Journal of Biological Chemistry, Vol . 266, pp . 8923-8931; C . S . Gibbs and M . J . Zoller (1991) Biochemistry, Vol . 30, p . 22} . This analysis indicated that the substrate binding parameters primarily depend on electrostatic interactions between a substrate or inhibitor and the C-subunit . Amino acid replacements that led to large perturbations in the electrostatic field are listed in the text . pKa shifts were also calculated for the substrate's phosphate accepting atom, the serine hydroxyl oxygen, when the active site ionizable residues were changed to structurally similar uncharged amino acids . The theoretical mutation of three active site residues caused large shifts in this parameter: E91Q, D166N, and D184N . The calculated pKa shifts for these mutants indicate that the rate of phosphotransfer should be markedly reduced in these cases . This prediction has been experimentally confirmed for the D166N mutant . The correlation between calculated electrostatic free energy changes and measured binding energy, and pKa shifts with phosphotransfer for C-subunit mutants were within experimental error of the measurements . The calculations of electrostatic energy and delta pKa have identified previously unconsidered active site residues in the mammalian C-subunit that contribute to binding energy and phosphotransfer.

Am J Hum Genet, 1996 Sep, 59(3), 625 - 32
Genetic and physical mapping of the Chediak-Higashi syndrome on chromosome 1q42-43; Barrat FJ et al.; The Chediak-Higashi syndrome (CHS) is a severe autosomal recessive condition, features of which are partial oculocutaneous albinism, increased susceptibility to infections, deficient natural killer cell activity, and the presence of large intracytoplasmic granulations in various cell types . Similar genetic disorders have been described in other species, including the beige mouse . On the basis of the hypothesis that the murine chromosome 13 region containing the beige locus was homologous to human chromosome 1, we have mapped the CHS locus to a 5-cM interval in chromosome segment 1q42.1-q42.2 . The highest LOD score was obtained with the marker D1S235 (Zmax = 5.38; theta = 0) . Haplo-type analysis enabled us to establish D1S2680 and D1S163, respectively, as the telomeric and the centromeric flanking markers . Multipoint linkage analysis confirms the localization of the CHS locus in this interval . Three YAC clones were found to cover the entire region in a conting established by YAC end-sequence characterization and sequence-tagged site mapping . The YAC contig contains all genetic markers that are nonrecombinant for the disease in the nine CHS families studied . This mapping confirms the previous hypothesis that the same gene defect causes CHS in human and beige pheno-type in mice and provides a genetic framework for the identification of candidate genes.

Mamm Genome, 1996 Sep, 7(9), 644 - 9
Molecular cloning and structural analysis of the functional mouse genomic XPG gene; Ludwig DL et al.; The mouse XPG gene is a homolog of the human DNA excision repair gene known to be defective in the hereditary sun-sensitive disorder xeroderma pigmentosum (group-G) . Defects in mouse XPG have been shown to directly affect the sensitivity of cultured cells to chemotherapy agents and may play a role in tumor cell drug resistance in vivo . A full-length cosmid clone of mouse XPG was isolated by complementation of the UV sensitivity and repair defect in CHO-UV135 cells . Exon mapping determined that the gene consisted of 15 exons within 32 kb of genomic DNA . Sequencing of intron-exon boundaries revealed that mouse XPG possesses a rare class of intron previously identified in only four other eukaryotic genes; it utilizes AT and AC dinucleotides instead of the expected GT and AG within the splice junctions . Promoter analysis determined that mouse XPG is expressed constitutively and probably initiates transcription from multiple start sites, yet, unlike the yeast homolog RAD2, we found no evidence that it is UVC inducible in cultured cells . Amino acid comparison with human XPG identified a highly conserved acidic region of homology not previously described.

DNA Res, 1996 Aug 31, 3(4), 239 - 55
Transcript map of the human chromosome 4p16.3 consisting of 627 cDNA clones derived from 1 Mb of the Huntington's disease locus; Hadano S et al.; Six hundred and twenty-seven cDNA clones from human brain cDNA libraries were characterized and integrated into a transcript map of the 1-Mb region on human chromosome 4p16.3 containing the Huntington's disease (HD) gene . Six hundred and seventy-two cDNA clones were obtained by a direct screening of the cDNA libraries, probing with pools of single copy microclones generated from the HD region specific yeast artificial chromosome (YAC)-DNA . So far, 93% of the obtained clones (627 cDNA clones) have been mapped onto the 1-Mb HD gene region by hybridization with HD region-specific cosmid, P1 and YAC clones . DNA sequence and expression analyses revealed that several cDNA clones might encode novel genes, some of which are situated within or close to the IT15, IT11, and alpha-adducin (ADD1) gene region, suggesting the presence of the overlapping genes in this region . This collection of cDNA clones will greatly facilitate the construction of the complete map of the transcripts in the HD region.

Science, 1996 Aug 30, 273(5279), 1239 - 41
A protein farnesyl transferase involved in abscisic acid signal transduction in Arabidopsis; Cutler S et al.; The hormone abscisic acid (ABA) modulates a variety of developmental processes and responses to environmental stress in higher plants . A collection of mutations, designated era, in Arabidopsis thaliana that confer an enhanced response to exogenous ABA includes mutations in the Era1 gene, which encodes the beta subunit of a protein farnesyl transferase . In yeast and mammalian systems, farnesyl transferases modify several signal transduction proteins for membrane localization . The era1 mutants suggest that a negative regulator of ABA sensitivity must be acted on by a farnesyl transferase to function.

J Biol Chem, 1996 Aug 30, 271(35), 21559 - 65
Conformations of the nucleotide and polypeptide binding domains of a cytosolic Hsp70 molecular chaperone are coupled; Fung KL et al.; 70-kDa heat shock protein (Hsp70) molecular chaperones are ATPases that participate in protein folding by regulating protein-protein interactions . ATP binds to the highly conserved amino-terminal domain, whereas polypeptides bind to the less conserved carboxyl-terminal domain . These domains are functionally coupled . Polypeptides were previously shown to dissociate from Hsp70s upon ATP binding and to stimulate ATPase activity . We probed the structure of the yeast cytosolic Hsp70 Ssa1p using limited proteolysis to determine whether the conformations of its nucleotide and polypeptide binding domains are also coupled . Ssa1p adopted three distinct conformations, nucleotide-free, ADP-dependent, and ATP-dependent . Complete conformational changes required K+ and Mg2+ . Using amino-terminal sequencing, ATP-agarose chromatography, and a carboxyl-terminal-specific antibody, we mapped the locations of the major proteolytic fragments . Nucleotides altered the conformations of both the nucleotide and polypeptide binding domains . Similarly, a polypeptide altered the conformations of both domains . These results indicate that the conformations of the nucleotide and polypeptide binding domains are coupled.

J Biol Chem, 1996 Aug 30, 271(35), 21365 - 74
A potential SH3 domain-binding site in the Crk SH2 domain; Anafi M et al.; The Src homology 2 (SH2) domain of the mammalian adaptor protein Crk-II contains a proline-rich insert, predicted to lie within an extended DE loop, which is dispensable for phosphopeptide binding . Using the yeast two-hybrid system, this region of the Crk-II SH2 domain was found to interact with a subset of SH3 domains, notably the Abl SH3 domain . Furthermore, this proline-rich insert was found to modify the efficiency with which Crk-II was phosphorylated by the p140(c-abl) tyrosine kinase . In vitro, the interaction of full-length non-phosphorylated Crk-II with a glutathione S-transferase-Abl SH3 domain fusion protein was very weak . However, phosphorylation of Crk-II on Tyr-221 which induces an intramolecular association with the SH2 domain, or addition of a phosphopeptide corresponding to the Crk-II Tyr-221 phosphorylation site, stimulated association of Crk-II with the Abl SH3 domain . NMR spectroscopic analysis showed that binding of the Tyr-221 phosphopeptide to the Crk SH2 domain induced a chemical shift change in Val-71, located in the proline-rich insert, indicative of a change in the structure of the proline-rich loop in response of Crk SH2-pTyr-221 interaction . These results suggest that the proline-rich insert in the Crk SH2 domain constitutes an SH3 domain-binding site that can be regulated by binding of a phosphopeptide ligand to the Crk SH2 domain.

Mol Gen Genet, 1996 Aug 27, 252(1-2), 87 - 92
Genetic analysis of two tomato mutants affected in the regulation of iron metabolism; Ling HQ et al.; Iron is one of the most important micronutrients for plants . Like other organisms, plants have developed active mechanisms for the acquisition of sufficient iron from the soil . Nevertheless, very little is known about the genetic mechanisms that control the active uptake . In tomato, two spontaneously derived mutants are available, which are defective in key steps that control this process . The recessive mutation chloronerva (chln) affects a gene which controls the synthesis of the non-protein amino acid nicotianamine (NA), a key component in the iron physiology of plants . The root system of the recessive mutant fer is unable to induce any of the characteristic responses to iron deficiency and iron uptake is thus completely blocked . We present a characterization of the double mutant, showing that the fer gene is epistatic over the chln gene and thus very likely to be one of the major genetic elements controlling iron physiology in tomato . In order to gain access to these two genes at the molecular level, both mutants were precisely mapped onto the high density RFLP map of tomato . The chln gene is located on chromosome 1 and the fer gene is on chromosome 6 of tomato . Using this high-resolution map, a chromosome walk has been started to isolate the fer gene by map-based cloning . The isolation of the fer gene will provide new insights into the molecular mechanisms of iron uptake control in plants.

FEBS Lett, 1996 Aug 26, 392(2), 179 - 83
Mammalian DNA cytosine-5 methyltransferase interacts with p23 protein; Zhang X et al.; In higher eukaryotic genomes, methylated cytosine residues (m(5)C) are distributed in heritable, cell-type-specific patterns, which are believed to be involved in the control of gene expression, developmental regulation and genomic imprinting . These methylation patterns are established and maintained by DNA cytosine-5 methyltransferase (MTase), a approximately 1500 amino acid enzyme containing a regulatory N-terminal domain and a catalytic C-terminal domain . The mechanism responsible for targeting MTase to particular genes is poorly understood and might possibly involve interactions with other proteins . In an effort to identify proteins that interact with the mammalian MTase, we used the yeast two-hybrid system with several different MTase domains as baits . Here we report an interaction between the C-terminal catalytic domain of the MTase and p23, a protein previously reported to associate with the progesterone receptor (PR) complex.

J Biol Chem, 1996 Aug 23, 271(34), 20432 - 7
Import of RNA into Leishmania mitochondria occurs through direct interaction with membrane-bound receptors; Mahapatra S et al.; Cytoplasmic tRNAs are imported into the kinetoplast mitochondrion of Leishmania, but the mechanism of import is unknown, particularly whether RNA is transferred as a ribonucleoprotein complex through the protein import pathway or by a distinct receptor-mediated mechanism . Using isolated mitochondria, it was shown that a small, importable RNA, which is structurally homologous to tRNA, binds rapidly, specifically, and with high affinity to the mitochondrial surface in the absence of soluble protein factors to form an import intermediate . Two classes of binding site of apparent Kd 0.3 and 10 n, respectively, were distinguished . tRNA from Leishmania, but not yeast, competitively inhibited the binding . Northwestern blot analysis revealed the presence of a 15-kDa RNA binding protein on the mitochondrial surface . Whereas receptor binding was resistant to heparin and KCl, internalization was sensitive to both reagents . These results are consistent with the presence of a direct mechanism of receptor-mediated RNA import on Leishmania mitochondria.

J Biotechnol, 1996 Aug 20, 49(1-3), 111 - 8
High speed centrifugal separator for rapid on-line sample clarification in biotechnology; Richardson P et al.; Sample clarification is a common operation in biochemical analytical methods for removing interfering or unwanted particulates from an analyte sample . Filtration provides one option for removal of particulates . However, in many cases the loss of soluble protein due to filter adsorption is unacceptable and an alternative must be sought . In this paper a microcentrifuge designed to automatically sample, spin, deliver supernatant to an analyser and wash out solids from the bowl is described . The performance of the system is assessed in terms of its clarification efficiency and the time required to achieve satisfactory clarification . Additionally, the effects of different protein precipitating agents on yeast homogenate samples separated using the microcentrifuge are studied where the system is used to deliver supernatant to a flow injection analyser . The paper demonstrates that the microcentrifuge may be used to separate rapidly such samples on a time scale between 10-60 s depending upon the type and size of sample and be successfully used as a component of an at-line monitoring system.

Biochemistry, 1996 Aug 20, 35(33), 10784 - 92
Mechanistic and structural contributions of critical surface and internal residues to cytochrome c electron transfer reactivity; Rafferty SP et al.; The influence of mutations in two conserved regions of yeast iso-1-cytochrome c believed to be critical to the mechanism of cytochrome c electron transfer reactions has been investigated . The variants Asn52Ala, Tyr67Phe, Ile75Met, and Thr78Gly involve perturbation of critical hydrogen-bonding interactions with an internal water molecule (Wat166) and have been studied in terms of their electrochemical properties and the kinetics with which they are reduced by Fe(EDTA)2- and oxidized by Co(phen)3(3+) . In parallel studies, the Co(phen)3(3+) oxidation kinetics of Tyr, Leu, Ile, Ala, Ser, and Gly variants of the phylogenetically conserved residue Phe82 have been studied and correlated with previous electrochemical and kinetic results . To assist mechanistic interpretation of these results, the three-dimensional structures of the Asn52Ala and Ile75Met ferrocytochrome c variants have been determined . The reduction potentials of the variants modified in the region of Wat166 were at least 33 mV (pH 6, 25 degrees C, and mu = 0.1 M) lower than that of the wild-type protein . Electron transfer reactivity of this family of variants in both the oxidation and reduction reactions was increased as much as 10-fold over that of the wild-type cytochrome . On the other hand, the reactivity of the position-82 variants in both oxidation and reduction depended on the structural characteristics of the oxidation-reduction reagent with which they reacted, and this reactivity was related to the nature of the residue at this position . These findings have been interpreted as demonstrating that the principal influence of modification at position-82 arises from changes in the nature of reactant-protein interaction at the surface of the protein and in maintaining the high reduction potential of the cytochrome while the principal influence of internal modifications near Wat166 results from alteration of the reorganization energy for the oxidation state-linked conformational change defined by crystallographic analysis of the wild-type protein.

FEBS Lett, 1996 Aug 19, 392(1), 63 - 5
Introns and protein revolution--an analysis of the exon/intron organisation of actin genes; Bagavathi S et al.; A catalogue of intron positions obtained from a large number of actin genes has been compiled with a view to understanding the possible origin of intervening sequences . Actins are ubiquitous proteins conserved in evolution and an analysis of their gene structures from various organisms has revealed that there may be at least 25 intron positions distributed at different positions in the coding regions . A comparison of intron positions from a wide range of organisms from that of yeast to human actins shows that introns could be more ancestral in origin . The conservation in the observed intron patterns within the different tissue types hints at a possible functional significance of introns in present day actin genes.

EMBO J, 1996 Aug 15, 15(16), 4330 - 43
Multiple interactions amongst floral homeotic MADS box proteins; Davies B et al.; Most known floral homeotic genes belong to the MADS box family and their products act in combination to specify floral organ identity by an unknown mechanism . We have used a yeast two-hybrid system to investigate the network of interactions between the Antirrhinum organ identity gene products . Selective heterodimerization is observed between MADS box factors . Exclusive interactions are detected between two factors, DEFICIENS (DEF) and GLOBOSA (GLO), previously known to heterodimerize and control development of petals and stamens . In contrast, a third factor, PLENA (PLE), which is required for reproductive organ development, can interact with the products of MADS box genes expressed at early, intermediate and late stages . We also demonstrate that heterodimerization of DEF and GLO requires the K box, a domain not found in non-plant MADS box factors, indicating that the plant MADS box factors may have different criteria for interaction . The association of PLENA and the temporally intermediate MADS box factors suggests that part of their function in mediating between the meristem and organ identity genes is accomplished through direct interaction . These data reveal an unexpectedly complex network of interactions between the factors controlling flower development and have implications for the determination of organ identity.

Genomics, 1996 Aug 15, 36(1), 192 - 6
A novel mouse mitochondrial voltage-dependent anion channel gene localizes to chromosome 8; Sampson MJ et al.; Voltage-dependent anion channels (VDACs) are small pore-forming channels found in the outer membrane of mitochondria . VDACs translocate adenine nucleotides and are the binding sites for several cytosolic kinases important in intermediary metabolism . Recently two human VDAC cDNAs (HVDAC1 and HVDAC2) were isolated, and possible orthologues of these genes have been isolated from mouse, bovine, and rat tissues . We report the isolation of a novel third VDAC cDNA from the mouse, designated MVDAC3 . The deduced MVDAC3 protein is approximately 70% identical to the previously isolated MVDAC1 and MVDAC2 proteins . The MVDAC3 gene was mapped by an interspecies backcross panel to mouse chromosome 8 . A database search using the mouse VDACs identified a second yeast VDAC-like gene that retains about 20% amino acid sequence identity with the mouse VDAC genes and 50% identity with the previously isolated yeast VDAC gene . The phylogenetic relationship of the eukaryotic VDAC genes is presented.

Genomics, 1996 Aug 15, 36(1), 185 - 8
Transcription elongation factor SII (TCEA) maps to human chromosome 3p22 --> p21.3; DiMarco SP et al.; Transcription elongation factors assist RNA polymerase II through transcriptional blockages . The human transcriptional elongation factor SII or Trascription Elongation Factor A (TCEA) releases RNA polymerase II from transcriptional arrest and is encoded by a 2.5-kb intronless gene . Using PCR primers, verified by RT-PCR to amplify the authentic, transcriptionally active SII gene, this locus was mapped to human chromosome 3 by examination of a human/rodent somatic cell hybrid panel . PCR analysis of somatic cell hybrids with chromosome 3 translocations and FISH studies utilizing a human YAC clone containing the SII gene further refine the map position of this locus to human chromosome 3p22 --> p21.3 . Since another elongation factor, SIII, has been implicated in human carcinogenesis and since the interval within which the human SII gene maps is frequently deleted in certain cancers, elongation factor SII may therefore be considered a candidate gene for human malignancies involving 3p22 --> p21.3.

Genomics, 1996 Aug 15, 36(1), 104 - 11
FISH-Mapped CEPH YACs spanning 0 to 46 cM on human chromosome 6; Bray-Ward P et al.; Seventy-six CEPH YACs were mapped by fluorescence in situ hybridization (FISH) to human metaphase chromosomes . These clones have been ordered from pter to 46 cM by combining the results of FISH with sequence-tagged site content mapping using data from the public databases . This created a minimal tiling path containing at least 37 Mb of human genomic DNA from 0 to 46 cM on chromosome 6 that contains up to four gaps not greater than 200 kb . These data provide an integration of the FLpter physical map values with cytogenetic band localization and markers on the genetic and radiation hybrid maps . We also assessed YAC chimerism and placed three additional Whitehead contigs (WC952, WC799, WC436) within the integrated map.

Genomics, 1996 Aug 15, 36(1), 70 - 85
A transcription map of the major histocompatibility complex (MHC) class I region; Gruen JR et al.; We have applied cDNA hybridization selection to nine YACs spanning 3 Mb of genomic DNA from a region centromeric to HLA-A to the histone cluster that lies telomeric to the human major histocompatibility complex (MHC) . In addition to Class I genes and pseudogenes, we describe over 63 genes and 23 additional expressed sequence tags distributed throughout the region . Many of the full-length genes belong to gene families . Prominent among these are a group of genes encoding proteins showing homology to the carboxyl-terminal sequences of butyrophilin and an additional group of zinc finger genes . We also detected several previously undefined genes that are specifically expressed in cells of the immune system, indicating a more complex role of the MHC in the immune response than has been appreciated.

Genomics, 1996 Aug 15, 36(1), 39 - 46
Physical analysis of the region deleted in the tw18 allele of the mouse tcl-4 complementation group; Barclay J et al.; We have generated a YAC contig of at least 3.3 Mb from the proximal region of In(17)4 of mouse chromosome 17 . This region corresponds to DNA lost in the gastrulation mutant tw18, which belongs to the tcl-4 complementation group . Our most proximal and distal probes lie within the deletion-3.3 Mb apart-indicating that we have not cloned the entire region . The deleted region is contained in a genetic interval of less than 1 cM, suggesting that some suppression of recombination must occur.

Anal Biochem, 1996 Aug 15, 240(1), 24 - 8
A general ribonuclease assay using methylene blue; Greiner-Stoeffele T et al.; Ribonuclease (RNase) activity has been assayed by monitoring the shift in the absorbance maximum of methylene blue upon intercalation into high-molecular-weight RNA . After preincubation of yeast RNA with methylene blue, the initial rate of hydrolysis can be followed spectrophotometrically at a wavelength of 688 nm . This allows enzyme kinetic studies of RNases with their probable native substrate, RNA, in place of the artificial ones, such as dinucleoside phosphates or cyclic monophosphates, currently used.

Virology, 1996 Aug 15, 222(2), 383 - 90
The Sendai virus V protein interacts with the NP protein to regulate viral genome RNA replication; Horikami SM et al.; The interactions of Sendai virus proteins required for viral RNA synthesis have been characterized both by the yeast two-hybrid system and through the use of glutathione S-transferase (gst)-viral fusion proteins synthesized in mammalian cells . Using the two-hybrid system we have confirmed the previously identified P-L (RNA polymerase), NPo-P (encapsidation substrate), and P-P complexes and now demonstrate NP-NP and NPo-V protein interactions . Expression of gstP and P proteins and binding to glutathione-Sepharose beads as a measure of complex formation confirmed the P-P interaction . The P-gstP binding occurred only on expression of the proteins in the same cell and was mapped to amino acids 345-411 . We also show that full-length and deletion gstV and gstW proteins bound NPo protein when these sets of proteins were coexpressed and have identified one required region from amino acids 78-316 . Neither gstV nor gstW bound NP assembled into nucleocapsids . Furthermore, both V and W proteins lacking the N-terminal 77 amino acids inhibited DI-H genome replication in vitro, showing the biological relevance of the remaining region . We propose that the specific inhibition of genome replication by V and W proteins occurs through interference with either the formation or the use of the NPo-P encapsidation substrate.

Genes Dev, 1996 Aug 15, 10(16), 2003 - 13
Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins; Alexandre C et al.; Members of the Hedgehog (Hh) family of secreted proteins have been identified recently as key signaling molecules that regulate a variety of inductive interactions central to the development of both Drosophila and vertebrates . Despite their widespread importance, the way in which Hh signals are transduced inside the cell remains poorly understood . The best candidate for a transcription factor that mediates Hh signaling in Drosophila is the product of the cubitus interruptus (ci) gene, a zinc finger protein that exhibits significant homology to protein products of the vertebrate GLI gene family . Here, we show that elevated levels of Ci are sufficient to activate patched (ptc) and other hh target genes, even in the absence of hh activity . We also show that Ci can function as a transcriptional activator in yeast and demonstrate that the zinc finger domain of the protein is sufficient for its target specificity . Finally, we identify sequences in the promoter region of the ptc gene, a primary target of Hh signaling, that are identical to the consensus-binding sequence of the GLI protein and are required for reporter gene expression in response to Hh activity . Taken together, our results strongly support the role for Ci as the transcriptional activator that mediates hh signaling.

Biochem J, 1996 Aug 15, 318 ( Pt 1), 287 - 90
The activity on double-stranded RNA of aggregates of ribonuclease A higher than dimers increases as a function of the size of the aggregates; Libonati M et al.; Stable bovine RNase A aggregates larger than dimers (identified as trimers, tetramers, pentamers and hexamers) were obtained by lyophilization of RNase A from 40-50% acetic acid solutions . The RNase activity of these aggregates was compared with that of monomeric RNase A on single- and double-stranded polyribonucleotides . Their activity toward poly(U) and yeast RNA slightly decreases as a function of the size of the aggregates . In contrast, their action on poly(A).poly(U) as substrate progressively increases from a relative activity of 1 for the RNase monomer to 10 for the hexamer . These results are discussed in the light of an already advanced hypothesis about a possible mechanism of RNase attack on double-stranded RNA.

Biochem J, 1996 Aug 15, 318 ( Pt 1), 247 - 53
Tyrosine phosphorylation and activation of a new mitogen-activated protein (MAP)-kinase cascade in human neutrophils stimulated with various agonists; Nahas N et al.; The presence of a novel 38 kDa protein that is tyrosine phosphorylated in human neutrophils, a terminally differentiated cell, upon stimulation of these cells with low concentrations of lipopolysaccharide (LPS) in combination with serum has been demonstrated . This 38 kDa protein was identified as the mammalian homologue of HOG1 in yeast, the p38 mitogen-activated protein (MAP) kinase . This conclusion is based on the experimental findings that anti-phosphotyrosine (anti-PY) antibody immunoprecipitates a 38 kDa protein that is recognized by anti-p38 MAP kinase antibody, and conversely, anti-p38 MAP kinase antibody immunoprecipitates a 38 kDa protein that can be recognized by anti-PY antibody . Moreover, this tyrosine phosphorylated protein is found associated entirely with the cytosol . It was also found that this p38 MAP kinase is activated following stimulation of these cells with low concentrations of LPS in combination with serum . This conclusion is based on three experimental findings . First, soluble fractions isolated from LPS-stimulated cells phosphorylate heat shock protein 27 (hsp27) in an in vitro assay, and this effect is not inhibited by protein kinase C and protein kinase A inhibitor peptides . This effect is similar to the effect produced by the commercially available phosphorylated and activated MAPKAP kinase-2 (MAP kinase activated protein kinase-2) . Secondly, a 27 kDa protein that aligns with a protein recognized by anti-hsp27 antibody is phosphorylated upon LPS stimulation of intact human neutrophils prelabelled with radioactive phosphate . Lastly, immune complex protein kinase assays, using {gamma-32P}ATP and activating transcription factor 2 (ATF2) as substrates, showed increased p38 MAP kinase activity from LPS-stimulated human neutrophils . The phosphorylation and activation of this p38 MAP kinase can be affected by both G-protein-coupled receptors such as platelet-activating factor (PAF) and non-G-protein-coupled receptors such as the cytokine-coupled receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor alpha (TNF-alpha) . The effect of low concentrations of PAF is greatly increased in cells pretreated with LPS . The tyrosine phosphorylation of the p38 MAP kinase is not restricted to stimuli that mediate their actions through membrane-associated receptors, but it can be affected by agents that bypass membrane-associated receptors such as the protein translation blocker anisomycin . While anisomycin is known to increase the tyrosine phosphorylation of the 54 kDa SAPK (stress-activated protein kinase), this is the first report that shows that anisomycin also tyrosine phosphorylates the p38 MAP kinase . Cytokine receptors that increase the tyrosine phosphorylation and activation of the erk1 and erk2 MAP kinases have less effect on this p38 MAP kinase than those that do not affect the erk1 and erk2 MAP kinases . The possible role of the p38 MAP kinase in the phosphorylation of cytosolic phospholipase A2 is discussed.

Oncogene, 1996 Aug 15, 13(4), 797 - 802
Characterization of t(11;14) translocation in mantle cell lymphoma by fluorescent in situ hybridization; Bigoni R et al.; Characterization of chromosome abnormalities in leukemia and lymphoma have contributed to the understanding of the molecular basis of these neoplastic diseases . In addition, specific chromosomal aberrations have acquired diagnostic or prognostic value . The t(11;14)(q13;q32) chromosome translocation has been detected in mantle cell lymphomas . However, possibly due to the limits of conventional cytogenetic analysis and the presence of different breakpoints at the molecular level, it is possible that the true percentage of association is underestimated . In our study, we used a yeast artificial chromosome, spanning the entire area where the rearrangements occur on chromosome 11q13, to detect the presence of translocations by fluorescent in situ hybridization experiments . We detected BCL-1 translocations in eight of eight patients with clinical and immunological features of mantle cell lymphoma, suggesting that the t(11;14) translocation is a critical event in the pathogenesis of MCL and may be a primary element for the diagnosis . Since this translocation is associated with poor prognosis, its detection may help to make a correct diagnosis as well as to evaluate residual disease, which is critical to plan a rational chemotherapy regimen.

Oncogene, 1996 Aug 15, 13(4), 721 - 9
Broadly expressed SNT-like proteins link FGF receptor stimulation to activators of Ras; Wang JK et al.; SNT was originally described as a approximately 90 kilodalton protein in neuronal precursor cells which bears affinity for the yeast cell cycle protein p13sucl and which undergoes rapid tyrosine phosphorylation following stimulation with growth factors which trigger terminal differentiation, but not by other growth factors which promote proliferation (Rabin et al., 1993) . We show here that similarly sized SNT-like proteins (SLPs) are expressed in fibroblast, myoblast, and lymphoid cell lines, and undergo robust tyrosine phosphorylation in response to several mitogenic ligands, including fibroblast growth factors (FGFs) . SLPs are tyrosine phosphorylated within 15 s of FGF stimulation, are predominantly membrane-associated, and are weakly associated with activated FGF receptor-1, suggesting that these proteins may be direct targets of the receptor kinase . Kinetic analysis of SLP phosphorylation and studies with serine/threonine kinase and phosphatase inhibitors suggest that SLPs are no larger than 70 000 kilodaltons, and that serine/threonine phosphorylation follows tyrosine phosphorylation to substantially retard gel electrophoretic mobility . SLPs are associated with the Grb-2 adaptor and are the major tyrosine phosphorylated proteins associated with the Ras guanine nucleotide exchange factor Sos in FGF-stimulated fibroblasts, suggesting that SLP-Grb2-Sos complexes modulate the activity of Ras proteins.

Biochim Biophys Acta, 1996 Aug 14, 1308(2), 97 - 102
Novel transcribed sequences represented in the complex genomic region 5q13; Morrison KE et al.; YACs from the complex repetitive human genomic region 5q13, spanning the spinal muscular atrophy (SMA) locus, have been searched for transcribed sequences using the method of End Ligation Coincident Sequence Cloning . Six transcripts (PT1-6) have been identified, three of which (PT4, PT5 and PT6) are novel . Five of these elements hybridise to multiple loci in 5q13, but PT5 is single copy and maps very close to markers that show linkage disequilibrium with SMA.

Biochem Biophys Res Commun, 1996 Aug 14, 225(2), 494 - 9
Differential mRNA expression of phospholipase D (PLD) isozymes during cAMP-induced differentiation in C6 glioma cells; Yoshimura S et al.; GTP gamma S-dependent phospholipase D (PLD) activity time-dependently increased during differentiation of rat C6 glioma cells to astrocytic phenotypes induced by dibutyryl cyclic AMP (dbcAMP)/theophylline . The changes in PLD mRNA level were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) method using the degenerate primers designed based on two conserved amino acid sequences in PLDs of human and yeast . The amplified three DNA fragments (tentatively termed as rPLDa, b, and c) contained the conserved regions present in PLDs of various organisms . RT-PCR using non-degenerate primers showed that rPLDa mRNA increased within 12h following treatment with dbcAMP, reaching a broad plateau and then returned to the initial level at 48h . In contrast, the level of rPLDb mRNA showed a concurrent decrease . rPLDc decreased in a time-dependent manner . These results suggest that the expression of PLD mRNAs are differentially regulated during differentiation in C6 glioma cells.

AIDS Res Hum Retroviruses, 1996 Aug 10, 12(12), 1151 - 9
Lack of in vivo effect of granulocyte-macrophage colony-stimulating factor on human immunodeficiency virus type 1; Scadden DT et al.; Neutropenia complicates HIV disease or its treatment in a large proportion of patients . Hematopoietic growth factor support has been tested in a number of clinical settings in HIV disease and has been demonstrated to be of benefit for specific parameters . One consideration regarding the use of hematopoietic growth factors in HIV disease is their potential effect on HIV viral burden, since alterations in HIV expression have been documented with certain cytokines in vitro . It has also been reported that some cytokines, notably GM-CSF, potentiate the antiviral properties of thymidine analogs such as zidovudine (AZT) in vitro . We tested these observations in vivo . Twelve HIV-positive patients with a CD4 cell count < or = 200/mm3 or HIV plasma viremia who were receiving a stable dose of zidovudine were enrolled into three dose cohorts of yeast-derived GM-CSF at 50, 125, or 250 micrograms/m2 daily by subcutaneous self-injection for 28 days . Measurements of HIV activity included serum acid-dissociated HIV p24 antigen levels, plasma and peripheral blood mononuclear cell (PBMC) limiting dilution HIV culture, and plasma HIV quantitative competitive polymerase chain reaction (PCR) . Serum and intracellular zidovudine levels were measured as well as hematologic, immunologic, and toxicity parameters . Virologic measures showed neither significant upregulation nor downregulation of serum acid-dissociated HIV p24 antigen, plasma and PBMC HIV culture, or PCR in association with GM-CSF administration . A trend toward increased intracellular AZT levels was noted, but this did not achieve statistical significance (p = 0.073) . CD4 and CD8 lymphocytes were essentially unaffected while absolute neutrophil counts increased with GM-CSF administration as expected . These data suggest that administration of GM-CSF does not perturb HIV activity or immunologic parameters in patients receiving AZT for advanced HIV disease . No potentiation of AZT antiviral effect was demonstrated.

J Biol Chem, 1996 Aug 9, 271(32), 19451 - 6
Overproduction, purification, and characterization of the XPC subunit of the human DNA repair excision nuclease; Reardon JT et al.; Xeroderma pigmentosum complementation group C gene (XPC) encodes a protein of 125 kDa which is present in a tight complex with a 58-kDa protein encoded by the human homolog of the yeast RAD23 gene, HHR23B (Masutani, C., Sugasawa, K., Yanagisawa, J., Sonoyama, T., Ui, M., Enomoto, T., Takio, K., Tanaka, K., van der Spek, P . J., Bootsma, D., Hoeijmakers, J . H . J., and Hanaoka, F.(1994) EMBO J . 13, 1831-1843) . The XPC-HHR23B complex is required for excision of thymine dimers from DNA in a human excision nuclease system reconstituted from purified proteins . In order to understand the role of the XPC-HHR23B complex in excision repair, we have overexpressed each subunit alone and the heterodimer in heterologous systems, purified them, and characterized their biochemical properties . We find that both XPC and the heterodimer bind DNA with high affinity and UV-damaged DNA with slightly higher preference . Surprisingly, we find that the XPC subunit alone is sufficient for reconstitution of the human excision nuclease and that the HHR23B subunit has no detectable effect on the excision activity of the reconstituted system.

J Biol Chem, 1996 Aug 9, 271(32), 19402 - 8
Prk, a cytokine-inducible human protein serine/threonine kinase whose expression appears to be down-regulated in lung carcinomas; Li B et al.; We have cloned and characterized a putative protein serine/threonine kinase termed prk through a combination of polymerase chain reaction and conventional cDNA library screening approaches . There are apparently two distinct domains within prk protein deduced from its nucleotide sequences . The amino-terminal portion has the feature of the catalytic domain of a serine/threonine kinase and shows strong homology to mouse fnk and other polo family kinases including mouse snk, human and murine plk, Drosophila polo, and yeast Cdc5 . The carboxyl-terminal portion, presumably the regulatory domain, shares extensive homology to mouse fnk . Northern blotting analyses reveal that prk expression is restricted to a very limited number of tissues with placenta, ovaries, and lung containing detectable amounts of prk mRNA . prk mRNA expression is also detected at a low level in the megakaryocytic cell line Dami, MO7e, and three brain glioma cell lines . In addition, refeeding of serum-deprived MO7e, Dami, and K562 cells of hematopoietic origin and GMOO637D of lung fibroblasts rapidly activates prk mRNA expression with its peak induction around 2 h after serum addition . prk gene activation by the serum requires no new protein synthesis . The recombinant cytokines such as interleukin-3 and thrombopoietin also activate prk mRNA expression in MO7e cells . Furthermore, a survey of RNAs isolated from the tumor and the uninvolved tissues from 18 lung cancer patients reveals that prk mRNA expression is significantly down-regulated in tumor tissues . Southern blotting analysis indicates that the prk gene is present in a single copy in the genome of tumors and normal cells . Taken together, these results suggest that prk expression may be restricted to proliferating cells and involved in the regulation of cell cycle progression . The molecular cloning of prk cDNA will facilitate the study of its biological role as well as its potential role in tumorigenesis.

J Biol Chem, 1996 Aug 9, 271(32), 19110 - 7
Mutations in a specific human serum albumin thyroxine binding site define the structural basis of familial dysalbuminemic hyperthyroxinemia; Petersen CE et al.; The familial dysalbuminemic hyperthyroxinemia (FDH) phenotype results from a natural human serum albumin (HSA) mutant with histidine instead of arginine at amino acid position 218 . This mutation results in an enhanced affinity for thyroxine . Site-directed mutagenesis and a yeast protein expression system were used to synthesize wild type HSA and FDH HSA as well as several other HSA mutants . Studies on the binding of thyroxine to these HSA species using equilibrium dialysis and quenching of tryptophan 214 fluorescence suggest that the FDH mutation affects a single thyroxine binding site located in the 2A subdomain of HSA . Site-directed mutagenesis of HSA and thyroxine analogs were used to obtain information about the mechanism of thyroxine binding to both wild type and FDH HSA . These studies suggest that the guanidino group of arginine at amino acid position 218 in wild type HSA is involved in an unfavorable binding interaction with the amino group of thyroxine, whereas histidine at amino acid position 218 in FDH HSA is involved in a favorable binding interaction with thyroxine . Neither arginine at amino acid position 222 nor tryptophan at amino acid position 214 appears to favorably influence the binding of thyroxine to wild type HSA.

J Theor Biol, 1996 Aug 7, 181(3), 293 - 8
Centromeric locations in karyotypes: a rule derived from the theory of branched polymers; Ostashevsky JY; It is believed that chromosomes occupy non-overlapping domains in the interphase nucleus, and that the nuclear volume can be divided into the interchromosomal space and the chromosome domains . Concentrations of various components (e.g., small ions) are different in these compartments . Since nuclear volume is twice as large in G2 as in G1 phase, V2/V1 (the G2/G1 ratio of total chromosomal volumes) must be two in order to keep the interchromosomal concentrations unchanged . The aim of this study is to test the 'V2/V1 = 2' hypothesis . It has been shown that G1-chromosomes behave as real flexible polymers . If a G2-chromosome behaves as a four-arm star-type branched polymer, then, according to polymer theory, its chromosome volume should depend on its centromere position . We calculated V2/V1 values for 40 karyotypes, from yeast to human, and 19 of them have V2/V1 = 2 +/- 10% . There are two types of exceptions from the 'V2/V1 = 2' rule: karyotypes with a large number of telocentric chromosomes (V2/V1 > 2), and karyotypes with a large number of metacentric chromosomes (V2/V1 < 2) . It has been observed in the literature that for all-telocentric karyotypes of mouse and Chinese muntjac, their chromosomes form branch-like structures by association of centromeres in clusters in G2 phase . When calculated for these temporary structures, V2/V1 decreases to two if the number of associated chromosomes per cluster is greater than or equal to five . This corresponds to a number of centromere clusters per nucleus less than or equal to 8-9 for mouse and Chinese muntjac, which is consistent with observation . For rye, all-metacentric karyotype, the calculated V2/V1 value increases to nearly two if B-chromosomes are taken into account.

Proc Natl Acad Sci U S A, 1996 Aug 6, 93(16), 8666 - 70
Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene; Mollereau C et al.; Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo . Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated . The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin . The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant . Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene . By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL . Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.

FEBS Lett, 1996 Aug 5, 391(1-2), 66 - 70
Interaction studies between the p21Cip1/Waf1 cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen (PCNA) by surface plasmon resonance; Knibiehler M et al.; The cyclin-dependent kinase (CDK) inhibitor p21Cip1 consists of two domains that interact with CDKs and proliferating cell nuclear antigen (PCNA), respectively . We have investigated the interaction between p21Cip1 and PCNA using surface plasmon resonance (SPR) technology and compared the results with those obtained from other sources such as the yeast two-hybrid system . Whilst other methods are only semi-quantitative, the SPR technique allowed us to determine the kinetic parameters of the interaction . The apparent equilibrium constant KD calculated for these kinetic parameters was 3.2 x 10(-7) M . We further demonstrate the use of SPR to study the interaction between mutant proteins and to determine their actual KD . The interaction between p21Cip1/PCNA is shown to be dependent upon the trimeric conformation of PCNA since a point mutant that abolishes PCNA-PCNA interaction also abolishes PCNA's interaction with p21Cip1 . Finally, we demonstrate that SPR can be used to characterise the interaction of p21Cip1 and PCNA in the presence of short competitive peptides.

J Biol Chem, 1996 Aug 2, 271(31), 18996 - 9000
Physical interaction between human RAD52 and RPA is required for homologous recombination in mammalian cells; Park MS et al.; The yeast RAD52 protein is essential for DNA double-strand break repair, and meiotic and mitotic recombination . RPA is a protein complex of three subunits (70, 34, and 11 kDa) that has been shown to be involved in DNA replication, nucleotide excision repair, and homologous recombination . Here, we demonstrate a physical interaction between human RAD52 and RPA in vivo and in vitro . In addition, the domain (amino acids 221-280) in RAD52 protein that mediates the interaction with the 34-kDa subunit of RPA was also determined . Overexpression of mutant RAD52 proteins lacking the interaction domain (amino acids 221-240, 241-260, and 261-280) failed to induce homologous recombination in monkey cells . We have previously shown that overexpression of human RAD52 induced homologous recombination in these cells . These results suggest that direct physical interactions between RAD52 and RPA are essential for homologous recombination in mammalian cells.

J Biol Chem, 1996 Aug 2, 271(31), 18859 - 68
Identification and cloning of centaurin-alpha . A novel phosphatidylinositol 3,4,5-trisphosphate-binding protein from rat brain; Hammonds-Odie LP et al.; Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP4), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-alpha . Binding specificity was determined using displacement of 1-O-{3H}(3-{4-benzoyldihydrocinnamidyl}propyl)-InsP4 photoaffinity labeling . Centaurin-alpha displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) (IC50 = 120 nM), whereas InsP4, PtdInsP2, and InsP3 bound with 5-, 12-, and >50-fold lower affinity, respectively . Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone . The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids . Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain . The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins . Given the specificity of binding and enrichment in brain, centaurin-alpha is a candidate PtdInsP3 receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain.

J Biol Chem, 1996 Aug 2, 271(31), 18405 - 12
A negative cofactor containing Dr1/p19 modulates transcription with TFIIA in a promoter-specific fashion; Kim J et al.; An activity that modulated the relative levels of transcription from the adenovirus major late promoter (MLP), and the immunoglobulin heavy chain mu promoter (mu) was purified as a 90-kDa factor . This factor is suggested to be a heterotetramer of two subunits: a 20-kDa polypeptide identical to the previously described Dr1/p19 and a novel 30-kDa polypeptide . The Dr1/p19 protein has been characterized as a repressor of transcription, and the 30-kDa protein is related to a recently identified yeast gene proposed to encode a repressor of transcription . The 90-kDa factor forms a complex with TATA-binding protein on DNA and at high concentrations of both factors protects over a 150-base pair region around the promoter from DNase I cleavage . The conformation of this complex as assayed by footprinting analysis is altered by the transcription factor TFIIA on the MLP but not on the mu promoter . Similarly, TFIIA reverses the repression of transcription by the 90-kDa factor on the MLP but not on the mu promoter . Thus, the interactions of TATA-binding protein, TFIIA, and the 90-kDa factor are promoter-specific.

Cell Struct Funct, 1996 Aug, 21(4), 237 - 43
Transcriptional and post-transcriptional regulation of pr22 (Op18) with proliferation control; Hosoya H et al.; The pr22 gene was isolated as a gene which is expressed in proliferating cells but not in cells which are differentiated or growth-arrested . When cells of the human monocytic cell line, U937, were differentiated into macrophages, transcription of pr22 was almost completely suppressed . Serum starvation resulted in the inhibition of transcription, although U937 failed to differentiate . In a culture synchronized with excess thymidine, mRNA of pr22 was detected at the G1/S boundary, with the level increasing in the S phase and decreasing in the G2 phase . The gene product, pr22 protein (Pr22) was found to be identical to Op18 as well as to a catastrophe factor . Genes homologous to pr22 were detected in the genome of mouse but not in that of yeast, or Drosophila . The 5' up-stream region of the genomic pr22 contained CpG islands but no TATA box at its appropriate position . About 20% of cell nuclei of normal human fibroblasts were stained in a speckled manner with a monoclonal antibody for C-terminal peptide of Pr22, and these cells were found to be in phases S and G2 . The mitotic apparatus was also strongly stained . By Western blot analysis, Pr22 was detected in the nuclear fraction but not in the cytoplasm . The level increased from middle S to G2 phase and remained high until the early G1 phase . N-terminal truncated Pr22 was also detected in these phases . These results suggest that Pr22 may have an additional role other than just functioning in association with microtubules.

Pharmacogenetics, 1996 Aug, 6(4), 341 - 9
The role of the CYP2C9-Leu359 allelic variant in the tolbutamide polymorphism; Sullivan-Klose TH et al.; Tolbutamide undergoes hydroxylation in humans via a cytochrome P450-mediated pathway . The primary P450 isozyme responsible for this metabolism is thought to be CYP2C9 . Population studies have indicated the existence of slow metabolizers of tolbutamide (approximately 1 in 500) suggesting a rare polymorphism associated with 2C9 . Several allelic variants of 2C9 have been identified; however, the effect of these allelic variations on metabolism in vivo is not established . In the present study, the coding regions, intron-exon junctions, and upstream region of CYP2C9 were amplified by PCR and sequenced in two slow metabolizers . One individual was homozygous for Leu359/Leu359 and the other individual was heterozygous for Arg144/Cys144 and for Ile359/Leu359 . No other genetic variations in 2C9 were detected in these individuals . PCR-RFLP tests showed that Arg144 Tyr358 Ile359 Gly417 is the principle CYP2C9 allele . Frequencies of the rarer Leu359 and Cys144 alleles were 0.06 and 0.08, respectively, in a Caucasian-American population and 0.005 and 0.01 respectively in African-Americans . The frequency of the Leu359 allele was 0.026 in Chinese-Taiwanese, but the Cys144 allele was not detected in this population . Studies in a recombinant yeast expression system showed that the Leu359 variant had the highest Km and the lowest Vmac for hydroxylation of tolbutamide of all the CYP2C9 allelic variants . This allelic variant also had the highest Km for the 7-hydroxylation of S-warfarin . The present data suggest that the incidence of the Leu359 allelic variant of CYP2C9 may account for the occurrence of poor metabolizers of tolbutamide.

Chromosome Res, 1996 Aug, 4(5), 372 - 83
Autosomal location of a new subtype of 1.688 satellite DNA of Drosophila melanogaster; Losada A et al.; During the screening of a Drosophila melanogaster YAC library with DNA from the minichromosome Dp(1;f)1187 we isolated a clone, yw20D5, which contains a new subtype of 1.688 satellite DNA . Although the sequences of several monomers subcloned from the YAC show a considerable variation in length, the derived consensus sequence is 356-bp long . This new subtype and the one constituted by the 353-bp repeats are both located on the left arm heterochromatin of chromosome 3, arranged in separate arrays . Despite their autosomal location, phylogenetic relationships among 1.688 satellite sequences suggest that they may have originated from the 359-bp repeats of the X chromosome heterochromatin . We have used the new 356-bp repeats to investigate whether sequences related to the 1.688 satellite are dispersed along the euchromatic arms of the autosomes in a similar way to that in which they are found along the X chromosome euchromatin.

Food Addit Contam, 1996 Aug-Sep, 13(6), 677 - 85
Co-production of aflatoxins and cyclopiazonic acid in isolates of Aspergillus flavus; Gqaleni N et al.; The distribution of total aflatoxin (AFT) and cyclopiazonic acid (CPA) between conidia and mycelial matrix was studied in five isolates of Aspergillus flavus Link cultured on maize grain for 20 days at 30 degrees C . Total aflatoxin and CPA production differed between the isolates with Aspergillus flavus F2R4FP 1-5 producing the most AFT (conidia-0.245 microgram/g; mycelial matrix-83 micrograms/g) and CPA (conidia-0.091 microgram/g; mycelial matrix-37 micrograms/g) . The production of AFT and CPA by this isolate was compared in liquid (malt-extract-yeast-extract-glucose) and solid substrates (yeast extract sucrose agar and maize) at 30 degrees C . The ratio of AFT:CPA in maize decreased from 16.6:1 to 1.6:1 between 7 and 21 days' incubation . In liquid culture and yeast extract sucrose agar, the ratios were reversed and ranged from 1:3 to 1:9 and 1:22 to 1:28, respectively, the greatest difference in both media occurring after 15 days.

Scand J Clin Lab Invest, 1996 Aug, 56(5), 461 - 70
Release of cytokines and proteases from human peripheral blood mononuclear and polymorphonuclear cells following phagocytosis and LPS stimulation; Ohlsson K et al.; Release and cellular contents of pro- and anti-inflammatory cytokines, neutrophilic elastase and secretory leukocyte proteinase inhibitor (SLPI) were measured with enzyme-linked immunosorbent assay in peripheral blood mono- and polymorphonuclear cells stimulated with preopsonized yeast cells or lipopolysaccharide . Tumour necrosis factor alpha (TNF alpha) was also measured with a bioassay . TNF alpha production and soluble TNF alpha receptor I (sTNF RI) were demonstrated in the environment of both cell populations . The bioassay indicated levels of TNF alpha far below those detected by ELISA . The overall secretion of cytokines and their inhibitors was found to favour an anti-inflammatory balance in the environment of the stimulated cells . The interleukin-1 receptor antagonist (IL1-ra), compared with interleukin-1 beta (IL-1 beta), dominated the secretions from both cell types with a 100- to 1000-fold excess respectively . Most of the translated IL-1 beta was not secreted but found associated with the cellular compartments . In contrast to lipopolysaccharide (LPS) stimulation, preopsonized yeast cells stimulated a massive release of elastase from neutrophil cells.

Virus Res, 1996 Aug, 43(2), 163 - 9
Expression of the major inner capsid protein of the epizootic haemorrhagic disease virus in baculovirus and potential diagnostic use; Nagesha HS et al.; The RNA 7 encoding the major capsid protein (VP7) of epizootic haemorrhagic disease virus (EHDV), Australian serotype 2 (strain CS439), was cloned and the complete nucleotide sequence was determined . The coding region contained 1047 nucleotides (nt) capable of encoding a predicted 349 amino acid (aa) polypeptide with a calculated molecular size of 38.087 kDa . When the VP7 gene was expressed in bacterial or yeast expression systems, the expression product showed weak or no reactivity with polyclonal antibodies to EHDV . Therefore, the expression of the VP7 gene in baculovirus was pursued . The expressed EHDV VP7 was similar in antigenicity to that of the native virus as revealed by its reactivity in ELISA with monoclonal antibody (MAb) specific to EHDV . Preliminary ELISA results indicated that the recombinant protein binds to EHDV antibodies in serum and that these antibodies block the binding of EHDV-specific MAb . The availability of a reliable EHDV recombinant VP7 could enhance our existing assay for detection of EHDV-specific antibodies.

Semin Pediatr Surg, 1996 Aug, 5(3), 182 - 90
Müllerian inhibiting substance as a model for the transforming growth factor-beta family: development of new treatment strategies; Shah PC et al.; The study of ligand receptor interactions and receptor function often requires multifaceted experimental approaches . In the course of studying the function and mechanism of action of mullerian inhibiting substance (MIS), we have used a wide range of molecular and cellular techniques . These have led to the identification, cloning, and characterization of the MIS receptors and of other receptors for the transforming growth factor-beta (TGF beta) family . This article describes the use of polymerase chain reaction (PCR) cloning to isolate candidate receptor genes, transfection and flow cytometry to study ligand binding, nonhomologous recombination targeted gene disruption (knockout) to analyze receptor function, and yeast genetics to identify other proteins that interact with the receptor complex . Together these techniques have led to the development of therapeutics and therapeutic strategies that are ready for clinical application.

Genome Res, 1996 Aug, 6(8), 747 - 60
Cloning of 559 potential exons of genes of human chromosome 21 by exon trapping; Chen H et al.; Chromosome 21 represents approximately 1% of the human genome, and its long arm has been estimated to contain 600-1000 genes . A dense linkage map and almost complete physical maps based on yeast artificial chromosomes (YACs) and cosmids have been developed . We have used exon trapping to identify portions of genes from randomly picked chromosome 21-specific cosmids, to contribute to the creation of the transcription (genic) map of this chromosome and the cloning of its genes . A total of 559 different sequences were identified after elimination of false-positive clones and repetitive elements . Among these, exons for 13 of the 30 known chromosome 21 genes have been "trapped." In addition, a considerable number of trapped sequences showed homologies to genes from other species and to human expressed sequence tags (ESTs) . One hundred thirty-three trapped sequences were mapped, and every one mapped back to chromosome 21 . We estimate that we have identified portions of up to approximately 40% of all genes on chromosome 21 . The genic map of chromosome 21 provides a valuable tool for the elucidation of function of the genes and will enhance our understanding of the pathophysiology of Down syndrome and other disorders of chromosome 21 genes.

Genome Res, 1996 Aug, 6(8), 688 - 701
Characterization of the mouse histone gene cluster on chromosome 13: 45 histone genes in three patches spread over 1Mb; Wang ZF et al.; The histone gene cluster on mouse chromosome 13 has been isolated and characterized . Using overlapping YAC clones containing histone genes from chromosome 13, a contig of approximately 2 Mb has been defined . It contains 45 histone genes, organized in three patches containing tightly clustered genes . An 80-kb patch (patch III) containing 12 histone genes is near one end of the contig, and a similar-sized patch (patch I) containing 15 histone genes is near the other end of the contig, located at least 500 kb from the central patch (patch II) of histone genes . The entire cluster contains six histone H1 genes, including the testis-specific histone H1t gene that maps to the middle of the cluster . All nine histone H3 genes in this cluster have been sequenced, and their level of expression determined . Each histone H3 gene is distinct, with five genes encoding the H3.2 protein subtype and four genes encoding the H3.1 protein . They are all expressed, with each histone H3 gene accounting for a small proportion of the total histone H3 mRNA.

Cell Growth Differ, 1996 Aug, 7(8), 1105 - 12
Cisplatin resistance and regulation of DNA repair in cAMP-dependent protein kinase mutants; Liu B et al.; Drug resistance in cancer poses a major problem to the success of chemotherapy . Increased resistance to the DNA-damaging chemotherapeutic drug cisplatin may be associated with a variety of factors including decreased drug accumulation, increased intracellular levels of thiols, and increased DNA repair . We have found that mutants of the Chinese hamster ovary (CHO) and the mouse adrenocortical carcinoma Y1 cells harboring a defective regulatory subunit (RI) of the cAMP-dependent protein kinase (PKA) exhibited increased resistance to cisplatin . These mutants are cross-resistant to other DNA-damaging chemotherapeutic agents, including bleomycin and melphalan . In addition, wild-type CHO cells transfected with and overexpressing the yeast phosphodiesterase gene or a dominant mutant Rl alpha subunit gene also displayed similar increased resistance to cisplatin . However, mutants with altered catalytic (C) subunits showed a sensitivity to cisplatin similar to the wild-type cells . Further analysis by gel shift assay using cisplatin-damaged DNA as probes and nuclear extracts derived from the Rl subunit mutants showed increased binding of nuclear factor(s) to the damaged DNA . In addition, a host cell reactivation assay of DNA repair, using a cisplatin-damaged reporter plasmid, detected enhanced capacity for repair of DNA lesions in the PKA mutants . These results suggest that DNA repair may be increased in the PKA mutants . We speculate that functional inactivation of PKA may result in increased DNA repair and the acquisition of resistance to DNA-damaging anticancer drugs in cancer.

J Ethnopharmacol, 1996 Aug, 53(2), 105 - 10
Investigation of antifungal and analgesic activities of extracts from Sium nodiflorum; Larhsini M et al.; Different extracts of aerial parts of Sium nodiflorum were examined for their antifungal activity against two groups of fungi: the growth of both yeast and mold was significantly inhibited . Analgesic activity study of these extracts was also carried out but showed no significant effect in mice on the writhing induced by acetic acid.

Hum Mol Genet, 1996 Aug, 5(8), 1139 - 48
Ott, a mouse X-linked multigene family expressed specifically during meiosis; Kerr SM et al.; The tissue expression patterns of 10 mouse testis cDNAs were analysed by RT-PCR to search for new mammalian meiotic genes . The homologue of the rat synaptonemal complex protein gene SCP1 is expressed in embryonic ovary, adult brain and testis . One novel gene is stringently testis specific and another is expressed exclusively in testis and embryonic ovary . The latter clone is not expressed in the testes of adult sex-reversed mice which lack germ cells, and therefore represents a meiosis-specific gene . It is part of a mouse multigene family, members of which are clustered and map genetically and physically to a single region of the X chromosome . We have named this family Ott (ovary testis transcribed) . Steady-state levels of a 2.3 kb polyadenylated Ott mRNA are high throughout meiotic prophase in the testis when the X chromosome is generally transcriptionally inactive . A second transcript of 1 kb is also detectable from 4 weeks of age onwards . The two mRNAs have different 3' ends and contain different protein coding information . At least seven Ott genes are transcribed specifically during meiosis and are predicted to encode "pioneer' proteins with an unusual structure, containing tandem arrays of a degenerate eight amino acid repeat . This work could lead to the identification of a human Ott homologue, which is likely to be X-linked and would provide a candidate locus for some cases of male infertility.

Biochem Mol Med, 1996 Aug, 58(2), 135 - 41
Fine mapping of the cystinosis gene using an integrated genetic and physical map of a region within human chromosome band 17p13; McDowell G et al.; The cystinosis gene has been reported to reside in a 3.1 cM region of chromosome 17p13 flanked by markers D17S1828 and D17S1798 . We created a yeast artificial chromosome (YAC) contig between these markers and report here an integrated genetic and physical map which will aid in the identification of other genes in this area . Using one pertinent YAC clone, 898A10, we identified new polymorphic markers in the cystinosis gene region . One such marker, D17S2167, was localized by radiation hybrid analysis to within 10.2 cR8000 of D17S1828 . Haplotype analysis in two separate informative families revealed recombination events which placed the cystinosis gene between markers D17S1828 and D17S2167, an area estimated to be 187-510 kb in size . This dramatic narrowing of the cystinosis gene region permits the creation of a P1 or cosmid contig across the area of interest . The ultimate cloning of the cystinosis gene should eventually reveal how a functional lysosomal transport protein is synthesized, targetted, processed, and integrated into the lysosomal membrane.

Genomics, 1996 Aug 1, 35(3), 431 - 40
A YAC, P1, and cosmid contig and 17 new polymorphic markers for the Werner syndrome region at 8p12-p21; Yu CE et al.; A yeast artificial chromosome (YAC), P1, and cosmid clone contig was constructed for the Werner syndrome (WRN) region of chromosome 8p12-p21 and used to clone a candidate gene for WRN . This region also possibly contains a familial breast cancer locus . The contig was initiated by isolating YACs for the glutathione reductase (GSR) gene and extended in either direction by walking techniques . Sequence-tagged site (STS) markers were generated from subclones of 2 GSR YACs and used to identify P1 and cosmid clones . Additional STSs were generated from P1 and cosmid clones and from potential expressed sequences identified by cDNA selection and exon amplification methods . The final contig was assembled by typing 17 YACs, 20 P1 clones, and 109 cosmids for 54 STS markers . The WRN region could be spanned by 2 nonchimeric YACs covering approximately 1.4 Mb . A P1/cosmid contig was established covering the core 700-800 kb of the WRN region . Fifteen new short tandem repeat polymorphisms and 2 biallelic polymorphic markers were identified and included as STSs in the contig . Analysis of these markers in Werner syndrome subjects demonstrates that the candidate WRN gene is in a region of linkage disequilibrium.

Curr Biol, 1996 Aug 1, 6(8), 1025 - 7
Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids; Makkerh JP et al.; Nuclear proteins contain information within their primary structures which causes them to accumulate selectively in the nucleus {1,2} by associating with the cytosolic receptor importin {3} . The alpha subunit of importin binds the nuclear localization signal (NLS), and the beta subunit docks at the nuclear pore complex . The NLS of the simian virus 40 large T-antigen (SV40 T-ag) is a single cluster of basic amino acids (PKKKRKV132; single-letter code, the basic amino acids are shown in bold; {4,5}), whereas the NLS of nucleoplasmin is bipartite . The nucleoplasmin NLS requires two essential clusters of basic amino acids, separated by a mutation-tolerant spacer (KRPAATKKAGQAKKKK171; {6} {7}) . A SwissProt database search shows that more than 50% of nuclear proteins contain a match to this consensus, and many NLSs have since been found to conform to this type of motif in yeast, plants and animals {8-10} . A different NLS (PAAKRVKLD) has been reported in the oncoprotein c-Myc, but it has received little attention because, unlike other known NLSs, only three of nine residues are basic {11}, and one residue is even acidic . Here, we report that constructs containing an inactive basic cluster downstream of the bipartite signal of nucleoplasmin can be directed to the nucleus by flanking them with specific neutral and acidic residues taken from the signal reported for c-Myc . Nuclear targeting by the single cluster KKKK is dependent on it being preceded by PAA and is stimulated if it is followed by the dipeptide LD . The relative positions of these elements are crucial to the function of these NLSs . All regions of the unconventional signal of c-Myc are functionally important . Contrary to conventional views, neutral and even acidic amino acids can play crucial roles in NLSs.

Planta Med, 1996 Aug, 62(4), 341 - 6
Detection and tissue distribution of anti-ulcer pectic polysaccharides from Bupleurum falcatum by polyclonal antibody; Sakurai MH et al.; Anti-sera against the "ramified" region (PG-1) of an anti-ulcer polysaccharide (bupleuran 2IIc), which was purified from the roots of Bupleurum falcatum L, were obtained by immunization of rabbits, and a polyclonal anti-bupleuran 2IIc/PG-1-antibody of the IgG class was purified by Protein G and "ramified" region (PG-1) immobilized affinity chromatographies . The antigenic specificity of anti-bupleuran 2IIc/PG-1-IgG was examined by a two-site sandwich ELISA which was developed as an improved method for microanalysis of bupleuran 2IIc using a biotinylated antibody . Another pectin from B . falcatum and anti-complementary pectins from Angelica acutilaba and Glycyrrhiza uralensis also showed significant reactivity to anti-bupleuran 2IIc/PG-1-IgG, although these reactivities were lower than that of bupleuran 2IIc . Other polysaccharides tested such as apple pectin, araban, yeast mannan, pullulan, etc., had negligible reactivity . The KDO-containing region and oligogalacturonides, which were obtained by endo-alpha-(1-->4)-polygalacturonase digestion of bupleuran 2IIc, were also not significantly recognized by anti-bupleuran 2IIc/PG-1-IgG . When bupleuran 2IIc was administered to the mice i.v., the polysaccharide disappeared from the circulation within 24 h and was mainly detected in the liver by the two-site sandwich ELISA . However the clearance of bupleuran 2IIc from the circulation was delayed by pretreatment with iota-carrageenan . When the crude polysaccharide fraction (BR-2), containing mainly bupleurans 2IIb and 2IIc from B.falcatum, was administrated orally to the mice, the polysaccharides were detected in the liver and Peyer's patch.

Plant Cell, 1996 Aug, 8(8), 1337 - 52
Nuclear import in permeabilized protoplasts from higher plants has unique features; Hicks GR et al.; The import of proteins into the nucleus is a poorly understood process that is thought to require soluble cytosolic factors in vertebrates and yeast . To test this model in plants and to identify components of the import apparatus, we developed a direct in vitro nuclear import assay by using tobacco protoplasts that were permeabilized without detergents such as digitonin or Triton X-100 . Substrates were imported specifically by a mechanism that required only guanine nucleotides . Moreover, in vitro import did not require exogenous cytosol . To investigate this novel finding, we isolated a full-length cDNA encoding an Arabidopsis homolog of vertebrate and yeast nuclear localization signal receptors and produced an affinity-purified antibody . The plant receptor was tightly associated with cellular components in permeabilized protoplasts, even in the presence of 0.1% Triton X-100, indicating that this factor and probably others were retained to an extent sufficient to support import . The lectin wheat germ agglutinin bound to the nucleus; however, it did not block translocation in our system, indicating that direct interaction with polysaccharide modifications at the nuclear pore complex was probably not essential for import in plants . Other features of in vitro import included reduced but significant import at low temperature.

Australas J Dermatol, 1996 Aug, 37(3), 137 - 8
Beta-1,3-D-glucan gel in the treatment of solar keratoses; Tong DW et al.; beta-1,3-D-glucans are yeast-derived carbohydrate polymers which have been shown to be potent immunoresponse modulators which promote the regression of certain tumours . To date there is no published data concerning the efficacy of topical beta-1,3-D-glucan in the treatment of solar keratoses . This randomized double-blind prospective pilot study of 20 patients was performed to investigate the efficacy and skin tolerance of beta-1,3-D-glucan gel versus placebo in the treatment of solar keratoses . The results of this study showed no significant benefit in using beta-1,3-D-glucan gel over placebo in reducing counts of solar keratoses . No adverse effects were reported by any patient at any stage of the trial.

J Virol, 1996 Aug, 70(8), 5266 - 71
An eight-nucleotide sequence in the potato virus X 3' untranslated region is required for both host protein binding and viral multiplication; Sriskanda VS et al.; Gel retardation and UV-cross-linking techniques were used to demonstrate that two tobacco proteins, with approximate molecular masses of 28 and 32 kDa, bind to a site within the 3' region of potato virus X (PVX) genomic RNA . The protein binding is specific, in that a 50-fold excess of unlabeled probe prevents formation of the complexes but no reduction is observed with a 2,000-fold molar excess of yeast tRNA . Complex formation is inhibited by poly(U) but is relatively unaffected by poly(A), poly(G), or poly(C-I) . PVX RNA-host protein complex formation occurs in vitro at salt concentrations up to 400 mM . Deletion mapping indicates that the proteins bind within the 3' untranslated region (UTR) of PVX genomic RNA and that an 8-nucleotide U-rich sequence (5'-UAUUUUCU) is required for the binding . Deletion of the 8-nucleotide U-rich region from the 3' UTR of a sensitive PVX reporter virus that carries the luciferase gene in place of the PVX coat protein gene results in a more than 70,000-fold reduction in luciferase expression in tobacco protoplasts . RNA probes carrying the sequence GCGC in place of the central four contiguous uridines of the 8-nucleotide U-rich motif fail to bind host protein at detectable levels, and the same mutation, when introduced into the PVX reporter virus, eliminates viral multiplication . Mutations of 1 or 2 nucleotides within the same four uridines reduced both binding of host proteins and replication of reporter virus . These results indicate that the 8-nucleotide U-rich motif within the PVX 3' UTR is important for some aspect of viral multiplication and suggest that host protein binding plays a role in the process.

J Virol, 1996 Aug, 70(8), 5025 - 34
Mutations in the carboxy-terminal domain of TBP affect the synthesis of human immunodeficiency virus type 1 full-length and short transcripts similarly; Pendergrast PS et al.; The human immunodeficiency virus type 1 promoter generates two types of RNA molecules, full-length transcripts and short transcripts . Synthesis of the short transcripts depends on the inducer of short transcripts (IST), an element located downstream of the start site . In the presence of the viral activator Tat, the synthesis of full-length transcripts is up-regulated while that of short transcripts is down-regulated . Full-length and short transcripts are probably generated by different types of transcription complexes . The first is IST independent, capable of efficient elongation, and up-regulated by Tat . The second is IST dependent, incapable of efficient elongation, and down-regulated by Tat . We have used an in vivo assay to assess the role of TBP in human immunodeficiency virus type I transcription and to test the effect of mutations in TBP on synthesis of full-length and short transcripts . We find that TBP bound to the TATA box is required for the synthesis of short and full-length transcripts as well as for Tat activation and that both yeast TBP and the carboxy-terminal domain of human TBP can replace full-length human TBP for these processes . Mutations in TBP affect the synthesis of short and full-length transcripts as well as Tat activation similarly, and these effects correlate with the previously described effects of these mutations on binding of TBP to the TBP-associated factor TAFII250 in vitro . Together, these results suggest that if short and full-length transcripts are generated by variant transcription complexes, these complexes use TBP similarly, probably as part of the TFIID complex.

Nucleic Acids Res, 1996 Aug 1, 24(15), 2990 - 7
Molecular cloning of a RNA binding protein, S1-1; Inoue A et al.; S1 proteins A-D constitute a nuclear protein family that are liberated rapidly in a set from chromatin by mild digestion with a DNA or RNA hydrolyzing enzyme . With an anti-S1-protein B antiserum that reacted with B2, C1 and D1, a cDNA clone, pS1-1, was obtained, which encoded a protein of 852 amino acids . The S1-1 protein, encoded within the cells by a mRNA of 3480 nt, was a novel protein and could be distinguished from the S1 proteins B, C and D by their amino acid sequences . The S-1-1 protein synthesized by in vitro translation bound to RNA homopolymers, with a preference for G and U polyribonucleotides and little for poly(A) . The protein contained two tandem RNP motifs and several intriguing sequences, such as a novel repeat of five octamers with a consensus sequence DP-S(Q/G)YYY and a potentially perfect amphipathic alpha-helix of five turns with basic and acidic amino acids positioned in an ordered way . The two RNP motif sequences were similar, although homologies were low, to the RNP motif sequences of yeast NSR1 protein, animal nucleolins, Drosophila hnRNP Al and tobacco chloroplast RNP precursor protein, suggesting a functional uniqueness of the S1-1 protein in RNA metabolism and also the evolution of its RNP motif structure before plants and animals diverged . These results indicate that the S1-1 protein encoded by the cDNA is a new class of RNA binding protein.

Nucleic Acids Res, 1996 Aug 1, 24(15), 2950 - 8
TBP binds the transcriptionally inactive TA5 sequence but the resulting complex is not efficiently recognised by TFIIB and TFIIA; Bernues J et al.; The binding of TBP (TFIID) to the TATA box has been considered to direct promoter recognition and pre-initiation complex formation because it is the first event leading to basal transcription by RNA polymerase II . Here, we analyse the binding of yeast TBP to a consensus TATAAA box and two point mutations, TAAAAA (inactive) and TATATA (active) . Despite the fact that the TAAAAA sequence does not support transcription in vitro, yeast TBP binds the three sequences showing, in this sense, only a limited sequence specificity . However, the TBP-TAAAAA complex cannot be recognised by other basal transcription factors, in particular by TFIIB . DNase I footprinting patterns of the TBP-TAAAAA complex are different from those observed in functional TBP-TATA box complexes, indicating that, most likely, it is a different spatial arrangement of the TBP-DNA complex that prevents formation of the TFIIB-TBP-TAAAAA complex, also seriously impairing entry of TFIIA to the complex . DNA deformability of the A/T-rich sequences appears to be an important determinant in the formation of a productive TBP-TATA complex . These results indicate that the transcriptional competence of A/T-rich sequences is determined not only by TBP binding, but also by the ability of other basal transcription factors to recognise the preformed TBP-DNA complexes.

Biochem J, 1996 Aug 1, 317 ( Pt 3), 633 - 41
Cell cycle regulation in Aspergillus by two protein kinases; Osmani SA et al.; Great progress has recently been made in our understanding of the regulation of the eukaryotic cell cycle, and the central role of cyclin-dependent kinases is now clear . In Aspergillus nidulans it has been established that a second class of cell-cycle-regulated protein kinases, typified by NIMA (encoded by the nimA gene), is also required for cell cycle progression into mitosis . Indeed, both p34cdc2/cyclin B and NIMA have to be correctly activated before mitosis can be initiated in this species, and p34cdc2/cyclin B plays a role in the mitosis-specific activation of NIMA . In addition, both kinases have to be proteolytically destroyed before mitosis can be completed . NIMA-related kinases may also regulate the cell cycle in other eukaryotes, as expression of NIMA can promote mitotic events in yeast, frog or human cells . Moreover, dominant-negative versions of NIMA can adversely affect the progression of human cells into mitosis, as they do in A . nidulans . The ability of NIMA to influence mitotic regulation in human and frog cells strongly suggests the existence of a NIMA pathway of mitotic regulation in higher eukaryotes . A growing number of NIMA-related kinases have been isolated from organisms ranging from fungi to humans, and some of these kinases are also cell-cycle-regulated . How NIMA-related kinases and cyclin-dependent kinases act in concert to promote cell cycle transitions is just beginning to be understood . This understanding is the key to a full knowledge of cell cycle regulation.

Bioessays, 1996 Aug, 18(8), 639 - 46
Promiscuity of fibroblast growth factor receptors; Green PJ et al.; Fibroblast growth factor receptors (FGFRs) have been implicated in many developmental and regenerative events, including axial organisation, mesodermal patterning, keratinocyte organisation and brain development . The consensus view that this reflects a role for one or other of the nine known members of the fibroblast growth factor family in these processes has recently been challenged by the suggestion that FGFRs might be directly activated by a much wider range of ligands, including heparan sulphate proteoglycans and neural cell adhesion molecules . In addition, two novel soluble ligands for FGFRs have been identified using yeast two-hybrid technology . Overall, the new findings suggest that in terms of ligand binding the FGFRs might be an even more promiscuous family of receptor tyrosine kinases than was already appreciated.

Mol Plant Microbe Interact, 1996 Aug, 9(6), 450 - 6
MPG1, a gene encoding a fungal hydrophobin of Magnaporthe grisea, is involved in surface recognition; Beckerman JL et al.; Upon encountering a leaf surface, emergent germ tubes from conidia of the rice blast fungus, Magnaporthe grisea, form infection structures called appressoria that allow direct penetration of plant cells . The MPG1 gene encodes a fungal hydrophobin of M . grisea that is expressed during development of aerial hyphae, conidia, and appressoria . Deletion of MPG1 reduces the efficiency of appressorium formation . We found that yeast extract repressed MPG1 expression in vitro and inhibited appressorium development of the rice pathogen, strain Guy11 . Appressorium formation of mpg1 mutants is rescued in trans by coinoculation with wild-type cells . MPG1 is required for efficient induction of appressoria in response to a host surface or highly hydrophobic artificial substrates . However, we identified several artificial substrates that can support efficient appressorium formation of mpg1 strains . This finding suggests that Mpg1p is not specifically required for appressorium formation, but is involved in the interaction with, and recognition of, the host surface . Additionally, a time window of competence to form appressoria was identified; the decision to form appressoria occurs approximately 6 to 8 h following conidial germination . After this critical time, cells are no longer able to form appressoria in response to inductive cues . These studies indicate that MPG1 hydrophobin is required for host recognition and that it acts as a morphogenetic signal for cellular differentiation.

Mol Cell Biol, 1996 Aug, 16(8), 4337 - 48
Isolation of a novel retinoic acid-responsive gene by selection of genomic fragments derived from CpG-island-enriched DNA; Shago M et al.; One of the primary goals in transcription factor research is the elucidation of the genetic networks controlled by a factor or by members of a family of closely related factors . The pleiotropic effects of retinoic acid (PA) in the developing and adult animal are mediated by ligand-inducible transcription factors (RA receptors {RARs} and retinoid X receptors {RXRs}) that belong to the superfamily of nuclear receptors . Regulatory regions of PA effector genes contain RAR and RXR binding sites (RAR elements {RAREs} and RXR elements {RXREs}) that generally consist of direct or everted repeats of the core half-site motif, (A/G)G(G/T)TCA . In order to identify novel genes regulated by RA, we devised a selection strategy based on the premise that regulatory regions of a large number of housekeeping and tissue-specific genes are embodied within CpG island DNA . In this method, referred to as CpG-selected and amplified binding, fragments derived from the CpG island fraction of the murine genome are selected by a gel mobility shift assay using in vitro-transcribed and -translated RXR-RAR . Multiple rounds of selection coupled with amplification of the fragments by PCR enabled us to clone a population of CG-rich fragments of which approximately one-fifth contained consensus RAREs or RXREs . Twelve genomic fragments containing novel response elements are described, and the transcription unit associated with one of them, NN-84AG, was characterized in detail . The mouse NN-84AG transcript is upregulated by RA in F9 embryonal carcinoma cells and is homologous to an expressed sequence tag (EST41159) derived from a human infant brain cDNA library . Cloning of the murine NN8-4AG genomic sequence places the RXRE in the proximity of the transcription initiation sites of the gene . Although sequence analysis indicates that the EST41159 gene product is novel, a region of amino acid identity with sequences of a yeast polypeptide of, as yet, unknown function and the Drosophila trithorax protein suggests the presence of an evolutionarily and functionally conserved domain . Our study demonstrates that transcription factor binding sites and corresponding regulated genes can be identified by selecting fragments derived from the CpG island fraction of the genome.

Mol Cell Biol, 1996 Aug, 16(8), 4207 - 14
Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay; Bogerd HP et al.; The Rex protein of human T-cell leukemia virus type 1, like the functionally equivalent Rev protein of human immunodeficiency virus type 1, contains a leucine-rich activation domain that specifically interacts with the human nucleoporin-like Rab/hRIP cofactor . Here, this Rex sequence is shown to function also as a protein nuclear export signal (NES) . Rex sequence libraries containing randomized forms of the activation domain/NES were screened for retention of the ability to bind Rab/hRIP by using the yeast two-hybrid assay . While the selected sequences differed widely in primary sequence, all were functional as Rex activation domains . In contrast, randomized sequences that failed to bind Rab/hRIP lacked Rex activity . The selected sequences included one with homology to the Rev activation domain/NES and a second that was similar to the NES found in the cellular protein kinase inhibitor alpha . A highly variant, yet fully active, activation domain sequence selected on the basis of Rab/hRIP binding retained full NES function even though this sequence preserved only a single leucine residue . In contrast, nonfunctional activation domain mutants that were unable to bind Rab/hRIP had also lost NES function . These data demonstrate that NES activity is a defining characteristic of the activation domains found in the Rev/Rex class of retroviral regulatory proteins and strongly support the hypothesis that the Rab/hRIP cofactor plays a critical role in mediating the biological activity of these NESs . In addition, these data suggest a consensus sequence for NESs of the Rev/Rex class.

Mol Cell Biol, 1996 Aug, 16(8), 4003 - 13
Determination of functional domains in the C subunit of the CCAAT-binding factor (CBF) necessary for formation of a CBF-DNA complex: CBF-B interacts simultaneously with both the CBF-A and CBF-C subunits to form a heterotrimeric CBF molecule; Kim IS et al.; The mammalian CCAAT-binding factor (CBF; also called NF-Y and CP1) is a heterotrimeric protein consisting of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and all of which are present in the CBF-DNA complex . In this study using cross-linking and immunoprecipitation methods, we first established that CBF-B interacts simultaneously with both subunits of the CBF-A-CBF-C heterodimer to form a heterotrimeric CBF molecule . We then performed a mutational analysis of CBF-C to define functional interactions with the other two CBF subunits and with DNA using several in vitro assays and an in vivo yeast two-hybrid system . Our experiments established that the evolutionarily conserved segment of CBF-C, which shows similarities with the histone-fold motif of histone H2A, was necessary for formation of the CBF-DNA complex . The domain of CBF-C which interacts with CBF-A included a large portion of this segment, one that corresponds to the segment of the histone-fold motif in H2A used for interaction with H2B . Two classes of interactions involved in formation of the CBF-A-CBF-C heterodimer were detected; one class, provided by residues in the middle of the interaction domain, was needed for formation of the CBF-A-CBF-C heterodimer . The other, provided by sequences flanking those of the first class was needed for stabilization of the heterodimer . Two separate domains were identified in the conserved segment of CBF-C for interaction with CBF-B; these were located on each side of the CBF-A interaction domain . Since our previous experiments identified a single CBF-B interaction domain in the histone-fold motif of CBF-A, we propose that a tridentate interaction domain in the CBF-A-CBF-C heterodimer interacts with the 21-amino-acid-long subunit interaction domain of CBF-B . Together with our previous mutational analysis of CBF-A (S . Sinha, I.-S . Kim, K.-Y . Sohn, B . de Crombrugghe, and S . N . Maity, Mol . Cell . Biol . 16:328-337, 1996), this study demonstrates that the histone fold-motifs of CBF-A and CBF-C interact with each other to form the CBF-A-CBF-C heterodimer and generate a hybrid surface which then interacts with CBF-B to form the heterotrimeric CBF molecule.

Chromosoma, 1996 Aug, 105(2), 70 - 81
Analysis of extrachromosomal structures containing human centromeric alphoid satellite DNA sequences in mouse cells; Taylor SS et al.; Yeast artificial chromosomes (YACs) spanning the centromeric region of the human Y chromosome were introduced into mouse LA-9 cells by spheroplast fusion in order to determine whether they would form mammalian artificial chromosomes . In about 50% of the cell lines generated, the YAC DNA was associated with circular extrachromosomal structures . These episomes were only present in a proportion of the cells, usually at high copy number, and were lost rapidly in the absence of selection . These observations suggest that, despite the presence of centromeric sequences, the structures were not segregating efficiently and thus were not forming artificial chromosomes . However, extrachromosomal structures containing alphoid DNA appeared cytogenetically smaller than those lacking it, as long as yeast DNA was also absent . This suggests that alphoid DNA can generate the condensed chromatin structure at the centromere.

FASEB J, 1996 Aug, 10(10), 1227 - 32
Dominant expression of a 1.3 Mb human Ig kappa locus replacing mouse light chain production; Zou X et al.; Expression studies of multigene families, such as the immunoglobulin (Ig) loci, are difficult because of their large size and the necessity to introduce germline configured regions into an animal . Antibody diversity from Ig gene miniloci is limited by the number of variable (V) region genes and the need for distal regulatory elements to control expression . Here, we show germline transfer into mice of a 1300 kb human Ig kappa light chain locus on a yeast artificial chromosome that resulted in early DNA rearrangement and highly efficient human light chain expression . The human locus was assembled from a 300 kb authentic region using contig extension by addition of cosmid multimers to supplement the variable gene cluster . This resulted in the addition of about 100 V region genes in germline configuration from different families . In transgenic animals with Ig kappa disruption, this large human kappa locus replaced the endogenous locus, and subsequent down-regulation of Ig lambda light chain contribution led to a dominant expression of the rearranged human genes . Contrary to expectation, rather than providing a solely selective advantage for ensuring repertoire formation controlled by the sheer number of introduced genes, the lambda/kappa ratio in serum appears to be the result of competition for early surface Ig expression maintained in the developing B cell.-Zou, X., Xian, J., Davies, N . P., Popov, A . V., Bruggemann, M . Dominant expression of a 1.3 Mb human Ig kappa locus replacing mouse light chain production.

J Cell Biol, 1996 Aug, 134(3), 699 - 714
emo-1, a Caenorhabditis elegans Sec61p gamma homologue, is required for oocyte development and ovulation; Iwasaki K et al.; emo-1(oz1) is a member of a class of hermaphrodite sterile mutations in Caenorhabditis elegans that produce endomitotic oocytes in the gonad arm . Oocytes in emo-1(oz1) mutants exhibit multiple defects during oogenesis . After meiotic maturation, ovulation fails, trapping oocytes in the gonad arm where they become endomitotic . emo-1 encodes a homologue of the Sec61p gamma subunit, a protein necessary for translocation of secretory and transmembrane proteins into the endoplasmic reticulum of yeast and mammalian cells . A putative emo-1 null mutation, oz151, displays embryonic lethality . The oz1 sterile mutation is a transposable element insertion into the emo-1 3' untranslated region that almost completely eliminates germline mRNA accumulation . Genetic mosaic analysis using the oz1 allele indicates that emo-1(+) expression in germ cells is required for fertility . The J67 monoclonal antibody, which recognizes an oocyte surface antigen (Strome, S . 1986 . In Gametogenesis and the Early Embryo . J.G . Gall, editor . Alan R . Liss, Inc., New York . 77-95.), does not stain oz1 oocytes, a finding consistent with defective protein transport in the mutant . We propose that the emo-1 gene product acts in the transport of secreted and transmembrane proteins in C . elegans oocytes, and is necessary for both oogenesis and the coupling of ovulation with meiotic maturation.

Clin Exp Immunol, 1996 Aug, 105(2), 205 - 12
Chronic mucocutaneous candidiasis . I . Altered antigen-stimulated IL-2, IL-4, IL-6 and interferon-gamma (IFN-gamma) production; Lilic D et al.; Patients with chronic mucocutaneous candidiasis (CMC) present with persistent infections with the opportunistic yeast Candida . Impaired cell-mediated responses to Candida have been documented in CMC patients, but the defect remains poorly understood . The importance of Th1 cytokines in resistance and Th2 in susceptibility to Candida infections has recently been demonstrated in murine models . In our studies we evaluated production of IL-2 and IFN-gamma (markers of Th1 type responses) as well as IL-4 and IL-6 (Th2 type markers) following stimulation with two kinds of Candida antigens (CAgs), polysaccharide antigens, tetanus toxoid and pokeweed mitogen . Our results demonstrate that CMC patients have impaired cytokine production upon in vitro stimulation with CAgs resulting in low or absent IL-2, increased IL-6 and either absent or increased IFN-gamma production . Cytokine production following stimulation by other antigens was unaltered . The overall cytokine-producing capacity assessed through mitogen stimulation was also intact . Addition of IFN-alpha or IFN-gamma to culture in an attempt to modify cytokine production did not have significant effects . Levels of soluble IL-6 receptors were not increased and could not account for increased IL-6 production . Our studies support the hypothesis that Candida antigens trigger a predominantly Th2 instead of a Th1 cytokine response in patients with CMC.

Hum Genet, 1996 Aug, 98(2), 125 - 8
A pericentric inversion of chromosome six in a patient with Peutz-Jeghers' syndrome and the use of FISH to localise the breakpoints on a genetic map; Markie D et al.; Karyotypic analysis in a patient with Peutz-Jeghers' syndrome demonstrated a pericentric inversion on chromosome 6 . Further investigation was undertaken using fluorescence in situ hybridisation (FISH) with yeast artificial chromosome clones selected to contain genetic markers from chromosome 6, and a probe for the centromeric alphoid repeat array . This analysis located one inversion breakpoint within the alphoid array, in a 1-cM interval between D6S257 and D6S402, and the other in a 4-cM interval between D6S403 and D6S311 . The oestrogen receptor gene locus (ESR) is excluded from the latter interval.

Clin Chem, 1996 Aug, 42(8 Pt 1), 1202 - 5
New enzymatic assay for calcium in serum; Kimura S et al.; We established a simple and rapid kinetic assay for measurement of calcium in serum by using urea amidolyase (EC 3.5.1.45) from yeast species . The method is based on inhibition of the enzyme by calcium . In the assay, we eliminated endogenous ammonium ion by use of glutamate dehydrogenase (GLDH; EC 1.4.1.4); then in the presence of urea amidolyase, urea, ATP, bicarbonate, magnesium, and potassium ions, ammonium ion production was inversely proportional to calcium ion concentration in serum . The concentration of ammonium ion formed was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH; we then monitored the change of absorbance at 340 nm . The within-run CVs of this method were 1.7-3.2% (n = 10) at 1.53-3.08 mmol/L, respectively . Day-to-day (total) CVs were 2.8-4.1% . Analytical recovery was 92-112% . The presence of other ions, ascorbic acid, reduced glutathione, bilirubin, hemoglobin, citrate, lipemic material, or human serum albumin did not affect this assay system . The correlation between values obtained with our method (y) and o-cresolphthalein complexone method (CPC) (x) was: y = 1.001x + 0.077 mmol/L (r = 0.949, Sy{symbol: see text}x = 0.079, n = 100); with the other enzymatic method (x) it was: y = 0.952x + 0.021 mmol/L (r = 0.955, Sy{symbol: see text}x = 0.074, n = 100) . The SEs for each method were: 0.025 mmol/L, our method; 0.023 mmol/L, CPC method; and 0.025 mmol/L, the other enzymatic method.

Nat Genet, 1996 Aug, 13(4), 458 - 60
A common region of 10p deleted in DiGeorge and velocardiofacial syndromes; Daw SC et al.; DiGeorge (DGS, MIM 188400) and velocardiofacial (VCFS, MIM 192430) syndromes may present many clinical problems including cardiac defects, hypoparathyroidism, T-cell immunodeficiency and facial dysmorphism . They are frequently associated with deletions within 22q11.2, but a number of cases have no detectable molecular defect of this region . A number of single case reports with deletions of 10p suggest genetic heterogeneity of DGS . Here we compare the regions of hemizygosity in four patients with terminal deletions of 10p (one patient diagnosed as having hypoparathyroidism and three as DGS) and one patient with a large interstitial deletion (diagnosed as VCFS) . Fluorescence in situ hybridization (FISH) analysis demonstrates that these patients have overlapping deletions at the 10p13/10p14 boundary . A YAC contig spanning the shortest region of deletion overlap (SRO) has been assembled, and allows the size of SRO to be approximated to 2 Mb . As with deletions of 22q11, phenotypes vary considerably between affected patients . These results strongly support the hypothesis that haploinsufficiency of a gene or genes within 10p (the DGSII locus) can cause the DGS/VCFS spectrum of malformation.

Nat Genet, 1996 Aug, 13(4), 409 - 16
X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein; Kere J et al.; Ectodermal dysplasias comprise over 150 syndromes of unknown pathogenesis . X-linked anhidrotic ectodermal dysplasia (EDA) is characterized by abnormal hair, teeth and sweat glands . We now describe the positional cloning of the gene mutated in EDA . Two exons, separated by a 200-kilobase intron, encode a predicted 135-residue transmembrane protein . The gene is disrupted in six patients with X;autosome translocations or submicroscopic deletions; nine patients had point mutations . The gene is expressed in keratinocytes, hair follicles, and sweat glands, and in other adult and fetal tissues . The predicted EDA protein may belong to a novel class with a role in epithelial-mesenchymal signalling.

Nat Genet, 1996 Aug, 13(4), 399 - 408
A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis; Feder JN et al.; Hereditary haemochromatosis (HH), which affects some 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals of Northern European descent, results in multi-organ dysfunction caused by increased iron deposition, and is treatable if detected early . Using linkage-disequilibrium and full haplotype analysis, we have identified a 250-kilobase region more than 3 megabases telomeric of the major histocompatibility complex (MHC) that is identical-by-descent in 85% of patient chromosomes . Within this region, we have identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations . One of these is predicted to inactivate this class of proteins and was found homozygous in 83% of 178 patients . A role of this gene in haemochromatosis is supported by the frequency and nature of the major mutation and prior studies implicating MHC class I-like proteins in iron metabolism.

Mamm Genome, 1996 Aug, 7(8), 575 - 9
Comparative mapping on the mouse X chromosome defines a myotubular myopathy equivalent region; de Gouyon B et al.; The gene for X-linked myotubular myopathy (MTM1) has been localized to a 300-kb critical region in human Xq28 between IDS and GABRA3 . As part of an effort to clone this gene, we developed a YAC contig on the mouse X Chromosome (Chr) which includes loci homologous to those within the human MTM1 critical region . The murine contig consists of 18 YACs and spans 2.5-3.0 Mb . We have aligned the human and murine physical maps by isolating conserved mouse genomic fragments, including CpG islands and trapped exons . We believe that the simultaneous isolation of genes from both mouse and human and continued comparative mapping will prove helpful in the eventual identification of MTM1 and other genes in the region.

EMBO J, 1996 Aug 1, 15(15), 4069 - 77
The var genes of Plasmodium falciparum are located in the subtelomeric region of most chromosomes; Rubio JP et al.; PfEMP1, a Plasmodium falciparum-encoded protein on the surface of infected erythrocytes is a ligand that mediates binding to receptors on endothelial cells . The PfEMP1 protein, which is encoded by the large var gene family, shows antigenic variation and changes in binding phenotype associated with alterations in antigenicity . We have constructed a yeast artificial chromosome contig of chromosome 12 from P . falciparum and show that var genes are arranged in four clusters; two lie amongst repetitive subtelomeric sequences and two occur in the more conserved central region . Analysis of parasite chromosomes by pulsed field gel electrophoresis (PFGE) demonstrates that most contain var genes and two-dimensional PFGE has shown that var genes are located at chromosome ends interspersed amongst repetitive sequences present in the subtelomeric complex . Analysis of a var gene located in the subtelomeric region of chromosome 12 has shown that it has close homologues at the opposite end of the chromosome and in the subtelomeric region of two other chromosomes . This suggests that recombination between heterologous chromosomes has occurred in the subtelomeric regions of these chromosomes . The subtelomeric location of var genes dispersed amongst repetitive sequences has important implications for generation of antigenic variants and novel cytoadherent specificities of this protein.

EMBO J, 1996 Aug 1, 15(15), 4061 - 8
SMC proteins constitute two subunits of the mammalian recombination complex RC-1; Jessberger R et al.; Recombination protein complex RC-1, purified from calf thymus nuclear extracts, catalyzes cell-free DNA strand transfer and repair of gaps and deletions through DNA recombination . DNA polymerase E, DNA ligase III and a DNA structure-specific endonuclease co-purify with the five polypeptide complex . Here we describe the identification of two hitherto unknown subunits of RC-1 . N-terminal amino acid sequences of the 160 and 130 kDa polypeptides display up to 100% identity to proteins of the structural maintenance of chromosomes (SMC) subfamilies 1 and 2 . SMC proteins are involved in mitotic chromosome segregation and condensation, as well as in certain DNA repair pathways in fission (rad18 gene) and budding (RHC18 gene) yeast . The assignment was substantiated by immuno-cross-reactivity of the RC-1 subunits with polyclonal antibodies specific for Xenopus laevis SMC proteins . These antibodies, and polyclonal antibodies directed against the bovine 160 and 130 kDa polypeptides, named BSMC1 and BSMC2 (bovine SMC), inhibited RC-1-mediated DNA transfer, indicating that the SMC proteins are necessary components of the reaction . Two independent assays revealed DNA reannealing activity of RC-1, which resides in its BSMC subunits, thereby demonstrating a novel function of these proteins . To our knowledge, this is the first evidence for the association of mammalian SMC proteins with a multiprotein complex harboring, among others, DNA recombination, DNA ligase and DNA polymerase activities.

EMBO J, 1996 Aug 1, 15(15), 3890 - 8
A two-component histidine kinase gene that functions in Dictyostelium development; Wang N et al.; A mutant which failed to complete development was isolated from a population of cells that had been subjected to insertional mutagenesis using restriction enzyme-mediated integration . The disrupted gene, dhkA, encodes the conserved motifs of a histidine kinase as well as the response regulator domain . It is likely that the histidine in DhkA is autophosphorylated and the phosphate passed to one or more response regulators . Such two-component systems function in a variety of bacterial signal transduction pathways and have been characterized recently in yeast and Arabidopsis . In Dictyostelium, we found that DhkA functions both in the regulation of prestalk gene expression and in the control of the terminal differentiation of prespore cells.

Biochim Biophys Acta, 1996 Jul 31, 1308(1), 58 - 66
Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver; Galande S et al.; A number of studies in yeast have shown that DNA topoisomerase II is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I . Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase II in chromosome disjunction . As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver . The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass . Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively . The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-free extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme . Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg(2+)-dependent manner . However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies . Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme . Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration . These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.

Cell, 1996 Jul 26, 86(2), 287 - 96
Pathway leading to correctly folded beta-tubulin; Tian G et al.; We describe the complete beta-tubulin folding pathway . Folding intermediates produced via ATP-dependent interaction with cytosolic chaperonin undergo a sequence of interactions with four proteins (cofactors A, D, E, and C) . The postchaperonin steps in the reaction cascade do not depend on ATP or GTP hydrolysis, although GTP plays a structural role in tubulin folding . Cofactors A and D function by capturing and stabilizing beta-tubulin in a quasi-native conformation . Cofactor E binds to the cofactor D-beta-tubulin complex; interaction with cofactor C then causes the release of beta-tubulin polypeptides that are committed to the native state . Sequence analysis identifies yeast homologs of cofactors D (cin1) and E (pac2), characterized by mutations that affect microtubule function.

Cell, 1996 Jul 26, 86(2), 263 - 74
SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box; Bai C et al.; We have identified the yeast and human homologs of the SKP1 gene as a suppressor of cdc4 mutants and as a cyclin F-binding protein . Skp1p indirectly binds cyclin A/Cdk2 through Skp2p, and directly binds Skp2p, cyclin F, and Cdc4p through a novel structural motif called the F-box . SKP1 is required for ubiquitin-mediated proteolysis of Cin2p, Clb5p, and the Cdk inhibitor Sic1p, and provides a link between these molecules and the proteolysis machinery . A large number of proteins contain the F-box motif and are thereby implicated in the ubiquitin pathway . Different skp1 mutants arrest cells in either G1 or G2, suggesting a connection between regulation of proteolysis in different stages of the cycle.

J Biol Chem, 1996 Jul 26, 271(30), 17597 - 600
A domain on the G protein beta subunit interacts with both adenylyl cyclase 2 and the muscarinic atrial potassium channel; Yan K et al.; The G protein betagamma complex modulates the function of a variety of effectors in biological signaling . However, the individual roles of the beta and gamma subunits in this interaction are unknown . Unlike in the case of the alpha subunit, domains on the betagamma complex that contact effectors have not yet been identified . We show here using the yeast two-hybrid system that the beta subunit and not the gamma subunit interacts with domains specific to adenylyl cyclase type 2 (AC2) and the muscarinic receptor-gated atrial inwardly rectifying potassium channel, GIRK1 . Different beta subunit types interact with these effector domains with different efficacies . Furthermore, an N-terminal fragment of 100 residues interacts with both these effector domains as effectively as the whole beta subunit . This domain includes the region where the beta subunit contacts with the alpha subunit in the crystal structure and may therefore explain the ability of the alpha subunit to shut off the activity of the betagamma complex.

J Biol Chem, 1996 Jul 26, 271(30), 17798 - 803
Regulation of 5'-AMP-activated protein kinase activity by the noncatalytic beta and gamma subunits; Dyck JR et al.; The mammalian 5'-AMP-activated protein kinase is a heterotrimer consisting of an alpha catalytic subunit and beta and gamma noncatalytic subunits, each of which is represented in a larger isoprotein family, related to the SNF1 kinase and its interacting proteins in yeast . In this study, we have used mammalian cell transfection to compare the activities of the two alpha subunit isoforms, alpha-1 and alpha-2, and to study the influence of the noncatalytic subunits on enzyme subunit association and activity . Expression of epitope-tagged protein subunits in COS7 cells indicates detectable but low level kinase activity for each of the two catalytic alpha subunits . Co-expression of alpha subunits with the beta or gamma subunits modestly increases kinase activity accompanied by the formation of alpha/beta or alpha/gamma heterodimers . Co-expression of all three subunits, however, is accompanied by a 50-110-fold increase in kinase activity with the formation of a heterotrimeric complex . In addition to binding of each noncatalytic subunit to the alpha subunit, the beta and gamma subunits bind to each other, likely resulting in a more stable heterotrimeric complex . The increase in kinase activity associated with expression of this heterotrimer is due both to an increase in enzyme-specific activity (units/enzyme mass) and to an apparent enhanced alpha subunit expression . Co-expression of a catalytically defective alpha subunit or the beta/gamma-binding COOH-terminal domain of the alpha subunit results in reduced heterotrimeric kinase activity . The synergistic positive regulatory roles for both the noncatalytic beta and gamma subunits of 5'-AMP-activated protein kinase contrasts with the Snf1p kinase, where only heterodimers of Snf1p and Snf4p seem to be required for maximum kinase activity.

Science, 1996 Jul 26, 273(5274), 488 - 90
Investigation of Ancient Egyptian Baking and Brewing Methods by Correlative Microscopy
Samuel D.
Ancient Egyptian methods of baking and brewing are investigated by optical and scanning electron microscopy of desiccated bread loaves and beer remains . The results suggest that current conceptions about ancient Egyptian bread and beer making should be modified . Bread was made not only with flour from raw grain, but sometimes also with malt and with yeast . Brewing blended cooked and uncooked malt with water; the mixture was strained free of husk before inoculation with yeast.

Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 8139 - 44
Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato; Lauter FR et al.; Root hairs as specialized epidermal cells represent part of the outermost interface between a plant and its soil environment . They make up to 70% of the root surface and, therefore, are likely to contribute significantly to nutrient uptake . To study uptake systems for mineral nitrogen, three genes homologous to Arabidopsis nitrate and ammonium transporters (AtNrt1 and AtAmt1) were isolated from a root hair-specific tomato cDNA library . Accumulation of LeNrt1-1, LeNrt1-2, and LeAmt1 transcripts was root-specific, with no detectable transcripts in stems or leaves . Expression was root cell type-specific and regulated by nitrogen availability . LeNrt1-2 mRNA accumulation was restricted to root hairs that had been exposed to nitrate . In contrast, LeNrt1-1 transcripts were detected in root hairs as well as other root tissues under all nitrogen treatments applied . Analogous to LeNrt1-1, the gene LeAmt1 was expressed under all nitrogen conditions tested, and root hair-specific mRNA accumulation was highest following exposure to ammonium . Expression of LeAMT1 in an ammonium uptake-deficient yeast strain restored growth on low ammonium medium, confirming its involvement in ammonium transport . Root hair specificity and characteristics of substrate regulation suggest an important role of the three genes in uptake of mineral nitrogen.

J Biol Chem, 1996 Jul 19, 271(29), 17002 - 5
A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance; Clemens JC et al.; We have used the yeast two-hybrid system to isolate a novel Drosophila adapter protein, which interacts with the Drosophila protein-tyrosine phosphatase (PTP) dPTP61F . Absence of this protein in Drosophila causes the mutant photoreceptor axon phenotype dreadlocks (dock) (Garrity, P . A., Rao, Y., Salecker, I., and Zipursky, S . L.(1996) Cell 85, 639-650) . Dock is similar to the mammalian oncoprotein Nck and contains three Src homology 3 (SH3) domains and one Src homology 2 (SH2) domain . The interaction of dPTP61F with Dock was confirmed in vivo by immune precipitation experiments . A sequence containing five PXXP motifs from the non-catalytic domain of the PTP is sufficient for interaction with Dock . This suggests that binding to the PTP is mediated by one or more of the SH3 domains of Dock . Immune precipitations of Dock also co-precipitate two tyrosine-phosphorylated proteins having molecular masses of 190 and 145 kDa . Interactions between Dock and these tyrosine-phosphorylated proteins are likely mediated by the Dock SH2 domain . These findings identify potential signal-transducing partners of Dock and propose a role for dPTP61F and the unidentified phosphoproteins in axonal guidance.

J Biol Chem, 1996 Jul 19, 271(29), 17161 - 6
Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-1beta, and MIP-1alpha; Raport CJ et al.; Chemokines affect leukocyte chemotactic and activation activities through specific G protein-coupled receptors . In an effort to map the closely linked CC chemokine receptor genes, we identified a novel chemokine receptor encoded 18 kilobase pairs downstream of the monocyte chemoattractant protein-1 (MCP-1) receptor (CCR2) gene on human chromosome 3p21 . The deduced amino acid sequence of this novel receptor, designated CCR5, is most similar to CCR2B, sharing 71% identical residues . Transfected cells expressing the receptor bind RANTES (regulated on activation normal T cell expressed), MIP-1beta, and MIP-1alpha with high affinity and generate inositol phosphates in response to these chemokines . This same combination of chemokines has recently been shown to potently inhibit human immunodeficiency virus replication in human peripheral blood leukocytes (Cocchi, F., DeVico, A . L., Garzino-Demo, A., Arya, S . K., Gallo, R . C., and Lusso, P.(1995) Science 270, 1811-1815) . CCR5 is expressed in lymphoid organs such as thymus and spleen, as well as in peripheral blood leukocytes, including macrophages and T cells, and is the first example of a human chemokine receptor that signals in response to MIP-1beta.

Nature, 1996 Jul 18, 382(6588), 262 - 5
Identification of the homologous beige and Chediak-Higashi syndrome genes; Barbosa MD et al.; Vesicular transport to and from the lysosome and late endosome is defective in patients with Chediak-Higashi syndrome (CHS) and in mutant beige (bg) mice . CHS and bg cells have giant, perinuclear vesicles with characteristics of late endosomes and lysosomes that arise from dysregulated homotypic fusion . CHS and bg lysosomes also exhibit compartmental missorting of proteins, such as elastase, glucuronidase and cathepsin G . Lyst, a candidate gene for bg, was identified by direct complementary DNA selection from a yeast artificial chromosome (YAC) clone containing a 650-kilobase segment of the bg-critical region on mouse chromosome 13 . Lyst is disrupted by a 5-kilobase deletion in bg mice, and Lyst messenger RNA is markedly reduced in bg homozygotes . The homologous human gene, LYST, is highly conserved with mouse Lyst, and contains a frame-shift mutation at nucleotides 117-118 of the coding domain in a CHS patient . Thus bg mice and human CHS patients have homologous disorders associated with Lyst mutations . Lyst encodes a protein with a carboxy-terminal prenylation motif and multiple potential phosphorylation sites . Lyst protein is predicted to form extended helical domains, and has a region of sequence similar to stathmin, a coiled-coil phosphoprotein thought to act as a relay integrating cellular signal response coupling.

Biochim Biophys Acta, 1996 Jul 17, 1307(3), 318 - 24
Cloning and characterization of complementary and genomic DNAs encoding the epsilon-subunit of rat translation initiation factor-2B; Flowers KM et al.; Eukaryotic initiation factor-2B (eIF-2B) is a guanine nucleotide-exchange protein involved in the recycling of eIF-2 during peptide-chain initiation . Regulation of eIF-2B activity occurs under a wide range of conditions by diverse mechanisms . To better understand the regulation of eIF-2B activity as well as the coordinate expression of its five subunits, we have begun to clone and characterize the cDNAs and genes encoding these proteins . In the present study, complementary and genomic DNAs encoding the epsilon-subunit of rat eIF-2B were cloned and characterized . The cDNA is 2517 bp in length, including a 30 nt poly(A) tail, and recognizes both 2.7 and 3.5 kb mRNA species on Northern blots of rat RNA . The cDNA contains a 2151 bp open reading frame encoding 716 amino acids producing a protein with a predicted molecular mass of 80 kDa . The derived amino acid sequence contains regions identical to three peptides obtained from bovine liver eIF-2B epsilon and is 31% identical to Gcd6, the putative yeast eIF-2B epsilon . Examination of the derived amino acid sequence of rat eIF-2B epsilon reveals phosphorylation site motifs for several protein kinases which have been implicated in regulation of guanine nucleotide exchange activity . The mRNA for eIF-2B epsilon is expressed to a similar extent in most rat tissues examined with the exception of testis, where its expression is approx, three-fold greater . We have also isolated and sequenced the coding and 5'-flanking region of the rat eIF-2B epsilon gene . The 16 exons encoding rat eIF-2B epsilon are contained within 9.5 kb of genomic DNA . Examination of the promoter region of the gene reveals a consensus binding site for the alpha-Pal transcription factor as well as possible cytokine-response elements and binding sites for testis-specific transcription factors.

Biochim Biophys Acta, 1996 Jul 17, 1307(3), 294 - 300
Sequence and expression pattern of an evolutionarily conserved transcript identified by gene trapping; Rijkers T et al.; We have isolated and analysed embryonic stem (ES) cell clones after electroporation with a gene trap vector . Clones were screened for changes in their lacZ reporter gene activity upon in vitro differentiation . The cDNA of one of the trapped transcripts, T10-2A2, was isolated and analysed in detail . Although not expressed constitutively in differentiating ES cells, the transcript was present in most organs of adult mice and widely expressed in midgestation mouse embryos . Zoo blot analysis indicated a conservation of this novel gene in yeast, rat and human.

Cancer Res, 1996 Jul 15, 56(14), 3165 - 7
Grb10: A new substrate of the insulin-like growth factor I receptor; Morrione A et al.; Using the yeast two-hybrid system, we have isolated a new substrate of the insulin-like growth factor I receptor (IGF-IR), identified as Grb10, a member of the family of SH2 domain proteins . With the help of several mutants of the IGF-IR, we have mapped the binding site of Grb10 between amino acids 1229 and 1245 of the receptor, a sequence that is dispensable for the mitogenic activity of the IGF-IR . Grb10 coprecipitates with the IGF-IR in cell lysates and is probably involved in the regulation of its activity.

Nucleic Acids Res, 1996 Jul 15, 24(14), 2813 - 20
E2A basic-helix-loop-helix transcription factors are negatively regulated by serum growth factors and by the Id3 protein; Loveys DA et al.; Id3, a member of the Id multigene family of dominant negative helix-loop-helix transcription factors, is induced sharply in murine fibroblasts by serum growth factors . To identify relevant targets of Id3 activity, the yeast two-hybrid system was used to identify proteins that dimerize with Id3 . Four murine cDNAs were identified in the screen, all of which encode helix-loop-helix proteins: E12, E47, ALF1 and Id4 . Co-immunoprecipitation assays confirm that Id3 interacts with E12, E47 and two alternative splice products of ALF1 in vitro . Id3 disrupts DNA binding by these proteins in vitro and blocks transcriptional activation by these factors in cultured murine cells . Additionally, Id3 shows evidence of interacting with the related proteins E2-2 and MyoD, but not c-Myc . These results suggest that Id3 can function as a general negative regulator of the basic-helix-loop-helix family of transcription factors exemplified by the 'E' proteins and MyoD . Although it was previously suspected that E2A is constitutively expressed, our data indicate that E2A is induced in quiescent fibroblasts, by growth factor withdrawal but not by contact inhibition of cell proliferation . These observations extend the role of Id3 in the functional antagonism of E2A-class transcription factors, and suggest that E2A proteins may mediate growth inhibition.

EMBO J, 1996 Jul 15, 15(14), 3702 - 12
Distinct domains of hTAFII100 are required for functional interaction with transcription factor TFIIF beta (RAP30) and incorporation into the TFIID complex; Dubrovskaya V et al.; TFIID is the DNA binding component of the RNA polymerase II transcriptional machinery and is composed of the TATA binding protein (TBP) and TBP-associated factors (TAFIIs) . Here we report the characterization of a new human TAF, hTAFII100, which is the human homologue of Drosophila TAFII80 and yeast TAFII90 . hTAFII100 interacts strongly with hTAFII250, hTAFII55 and hTAFII28, less with hTAFII20 and hTAFII18, weakly with TBP and not at all with delta NTAFII135 and hTAFII30 . Deletion analysis revealed that the C-terminal half of hTAFII100, which contains six WD-40 repeats, is not required for incorporation into the TFIID complex . Our results suggest that hTAFII100 can be divided into two domains, the N-terminal region responsible for interactions within the TFIID complex and the C-terminal WD repeat-containing half responsible for interactions between hTAFII100 and other factors . An anti-hTAFII100 antibody, raised against a C-terminal epitope, selectively inhibited basal TFIID-dependent in vitro transcription and the specific interaction between hTAFII100 and the 30 kDa subunit of TFIIF (RAP30) . We demonstrate that the hTAFII100-TFIIF interaction supports pre-initiation complex formation in the presence of TFIID . Thus, this is the first demonstration that a TAFII functionally interacts with a basal transcription factor in vitro.

Cancer Genet Cytogenet, 1996 Jul 15, 89(2), 153 - 6
Detection of the breakpoint cluster region-ABL fusion in chronic myeloid leukemia with variant Philadelphia chromosome translocations by in situ hybridization; Tosi S et al.; Fluorescence in situ hybridization (FISH) technique has been successfully used to detect the BCR-ABL gene fusion in chronic myeloid leukemia (CML) with the classic form of the Philadelphia chromosome (Ph) . We applied FISH to study three CML patients showing variant Ph chromosome (either complex or simple type) . The results demonstrate that the use of a yeast artificial chromosome (YAC)-derived probe (D107F9) and a cosmid probe (cos-abl 8), specific for BCR and ABL genes respectively, allows also the detection of the BCR-ABL fusion in CML patients with variant Ph.

EMBO J, 1996 Jul 15, 15(14), 3590 - 8
Newly assembled cyclin B-cdc2 kinase is required to suppress DNA replication between meiosis I and meiosis II in starfish oocytes; Picard A et al.; Micro-injection of catalytically inactive GST-cdc2-K33R or GST-cdk2-K33R fusion proteins, each of which efficiently titrates cyclin B in oocytes and prevents assembly of cyclin B-cdc2 complexes, readily induces premature DNA replication in starfish oocytes after emission of the first polar body . Moreover, partial ablation of cyclin B mRNA by micro-injection of antisense oligonucleotides facilitates premature DNA replication induced by the dominant-negative cdc2 and cdk2 mutant proteins . We thus propose that enhanced translation of cyclin B after GVBD, a universal feature of oocyte maturation in the animal kingdom, and subsequent assembly of cyclin B-cdc2 complexes, are part of the checkpoint that prevents DNA replication in the oocyte after emission of the first polar body . MAPK inactivation is neither required for premature DNA replication after the first meiotic cell cycle nor for DNA replication after completion of meiotic maturation . However, micro-injection of a N-terminally truncated form of the budding yeast STE11 protein, that constitutively maintains MAPK active after the second meiotic cleavage, prevents fertilized eggs from proceeding into embryogenesis, and arrests them at G2, as is the case in unfertilized eggs that cannot inactivate MAPK after the second meiotic cleavage . We thus propose that MAPK functions in meiotic maturation by preventing unfertilized eggs from proceeding into parthenogenetic development.

Genomics, 1996 Jul 15, 35(2), 392 - 6
cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/myeloid leukemia factor 2 (MLF2); Kuefer MU et al.; A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia . Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity) . In contrast to the tissue-restricted expression pattern of MLF1, the MLF2 messenger RNA is expressed ubiquitously . The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal region frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage . In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9 . Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements.

Genomics, 1996 Jul 15, 35(2), 380 - 2
The MAS proto-oncogene is not imprinted in humans; Riesewijk AM et al.; Recently it was shown that the murine Mas gene, which is located less than 300 kb from the imprinted Igf2r gene, is also imprinted in Day 11.5 embryos with expression exclusively from the paternal allele . We have assigned the human MAS gene to chromosomal bands 6q25.3-q26 in close proximity to the IGF2R gene . In contrast to its murine homologue, the human IGF2R gene is not imprinted . By making use of a novel intragenic polymorphism, we have studied the expression of the MAS gene in three heterozygous human fetuses . In all tissues examined, including tongue, biallelic expression of the MAS gene was observed . Hence both MAS and the neighboring IGF2R gene are not imprinted in humans.

Genomics, 1996 Jul 15, 35(2), 367 - 71
The active gene that encodes human high mobility group 1 protein (HMG1) contains introns and maps to chromosome 13; Ferrari S et al.; The human genome contains a large number of sequences related to the cDNA for High Mobility Group 1 protein (HMG1), which so far has hampered the cloning and mapping of the active HMG1 gene . We show that the human HMG1 gene contains introns, while the HMG1-related sequences do not and most likely are retrotransposed pseudogenes . We identified eight YACs from the ICI and CEPH libraries that contain the human HMG1 gene . The HMG1 gene is similar in structure to the previously characterized murine homologue and maps to human chromosome 13 band q12, as determined by in situ hybridization . The mouse Hmg1 gene maps to the telomeric region of murine Chromosome 5, which is syntenic to the human 13q12 band.

Genomics, 1996 Jul 15, 35(2), 353 - 60
A 500-kb physical map and contig from the Harvey ras-1 gene to the 11p telomere; Russell MW et al.; A contiguous physical map was constructed from the Harvey ras-1 (HRAS1) gene to the 11p telomere . The contig spans approximately 500 kb and is minimally composed of a telomere-containing YAC and P1 and cosmid clones . Included in the contig are 11 sequence-tagged sites derived from P1 and cosmid ends . Three genes were placed on the contig in the following order: telomere-ribonuclease/angiogenin inhibitor (RNH)-Harvey ras-1 (HRAS1)-HRAS1-related complex (HRC) . Two novel tetranucleotide repeats (heterozygosity of 66 and 68%) and a complex CA repeat (heterozygosity of 78%) were isolated and characterized.

Genomics, 1996 Jul 15, 35(2), 338 - 45
Cosmids map two incontinentia pigmenti type 1 (IP1) translocation breakpoints to a 180-kb region within a 1.2-Mb YAC contig; Gorski JL et al.; Incontinentia pigmenti (IP) is an X-linked dominant disorder of neuroectodermal development . Based on the observation of six unrelated females with clinical features of nonfamilial IP with constitutional de novo reciprocal X;autosome translocations, a putative incontinentia pigmenti type 1 locus (IP1; MIM No . 308300) was localized to region Xp11.21 . Using available regional DNA markers, we constructed a yeast artificial chromosome (YAC) contig that contained 1.2 Mb of distal Xp11.21 and spanned two IP1 X-chromosomal breakpoints . This contig was used to generate a detailed molecular map of the region and identify three regional CpG islands . YAC-derived cosmids were used to clone and map the IP1 breakpoints to a 180-kb interval that was flanked by DNA markers DXS705 and DXS741 . The physical map and genomic clones should facilitate the isolation and characterization of transcripts associated with the IP1 translocation breakpoints.

Genomics, 1996 Jul 15, 35(2), 289 - 98
Cloning, chromosomal localization, physical mapping, and genomic characterization of HKR3; Maris JM et al.; The Kruppel-type zinc finger proteins are members of a conserved family of transcription factors that are important in developmental regulation . Altered expression of several of these proteins has been implicated in human diseases, including cancer . We report the cloning, mapping, and characterization of the zinc finger gene Human Kruppel-Related 3 (HKR3) . Genomic clones of HKR3 were isolated from a P1 library and localized to human chromosome subband 1p36.3 by human-rodent somatic cell hybrid mapping and fluorescence in situ hybridization . The gene was physically mapped to within 40 kb of D1S214 by YAC content and long-range restriction mapping . HKR3 spans 9.5 kb of genomic DNA and is contained in 11 exons . Sequencing defined each of the exon/intron splice site junctions and identified a CpG island in the 5' region of the gene . HKR3 is ubiquitously expressed in human tissues as at least two major transcripts, the shorter of which excludes a conserved finger-associated box and a putative acidic activation domain contained in the full-length transcript . HKR3 is a novel zinc finger gene that maps to a region of the genome commonly rearranged or deleted in human cancers.

Cell, 1996 Jul 12, 86(1), 71 - 82
Influence of PAX6 gene dosage on development: overexpression causes severe eye abnormalities; Schedl A et al.; Aniridia in man and Small eye in mice are semidominant developmental disorders caused by mutations within the paired box gene PAX6 . Whereas heterozygotes suffer from iris hypoplasia, homozygous mice lack eyes and nasal cavities and exhibit brain abnormalities . To investigate the role of gene dosage in more detail, we have generated yeast artificial chromosome transgenic mice carrying the human PAX6 locus . When crossed onto the Small eye background, the transgene rescues the mutant phenotype . Strikingly, mice carrying multiple copies on a wild-type background show specific developmental abnormalities of the eye, but not of other tissues expressing the gene . Thus, at least five different eye phenotypes are associated with changes in PAX6 expression . We provide evidence that not only reduced, but also increased levels of transcriptional regulators can cause developmental defects.

J Biol Chem, 1996 Jul 12, 271(28), 16952 - 61
The in vitro generation of post-Golgi vesicles carrying viral envelope glycoproteins requires an ARF-like GTP-binding protein and a protein kinase C associated with the Golgi apparatus; Simon JP et al.; We have developed a system that recreates in vitro the generation of post-Golgi vesicles from an isolated Golgi fraction prepared from vesicular stomatitis virus- or influenza virus-infected Madin-Darby canine kidney or HepG2 cells . In this system, vesicle generation is temperature- and ATP-dependent and requires a supply of cytosolic proteins, including an N-ethylmaleimide-sensitive factor distinct from NSF . Cytosolic proteins obtained from yeast were as effective as mammalian cytosolic proteins in supporting vesicle formation and had the same requirements . The vesicles produced (50-80 nm in diameter) are depleted of the trans Golgi marker sialyltransferase, contain the viral glycoprotein molecules with their cytoplasmic tails exposed, and do not show an easily recognizable protein coat . Vesicle generation was inhibited by brefeldin A, which indicates that it requires the activation of an Arf-like GTP-binding protein that promotes assembly of a vesicle coat . Vesicles formed in the presence of the nonhydrolyzable GTP analogue guanosine 5'-3-O-(thio)triphosphate retained a nonclathrin protein coat resembling that of COP-coated vesicles, and sedimented more rapidly in a sucrose gradient than the uncoated ones generated in its absence . This indicates that GTP hydrolysis is not required for vesicle generation but that it is for vesicle uncoating . The activity of a Golgi-associated protein kinase C (PKC) was found to be necessary for the release of post-Golgi vesicles, as indicated by the capacity of a variety of inhibitors and antibodies to PKC to suppress it, as well as by the stimulatory effect of the PKC activator 12-O-tetradecanoylphorbol-13-acetate.

Exp Cell Res, 1996 Jul 10, 226(1), 11 - 9
Localization of the N-terminus of SCP1 to the central element of the synaptonemal complex and evidence for direct interactions between the N-termini of SCP1 molecules organized head-to-head; Liu JG et al.; The synaptonemal complex (SC) is a meiosis-specific, tripartite structure essential for synapsis of homologous chromosomes; it contains a central element positioned between two lateral elements and transversal filaments connecting the lateral elements . In mammals, a major constituent of the transversal filament is known: the SCP1 protein . It contains a long central coiled-coil motif and the molecules are probably organized as dimers, each forming a coiled-coil fiber . We have now developed a new sensitive procedure for immunoelectron microscopy of synaptonemal complex proteins and determined the exact localization of the two nonhelical ends of the SCP1 protein within the mouse synaptonemal complex . We found that the N-terminal end of the SCP1 protein is located within the central element of the synaptonemal complex, whereas the C-terminal end is close to or within the lateral element of the synaptonemal complex . This result supports the notion that SCP1 is an extended filamentous protein and that the two molecules of the putative SCP1 dimer are likely to have the same polarity . The observation that the N-termini are confined to the central element indicated that SCP1 dimers, anchored in opposite lateral elements, could establish contact with each other in the central element via their N-termini . To test this possibility we used the yeast two-hybrid system and found that the N-terminal end of the SCP1 protein indeed strongly interacted with itself, but not with other protein domains tested . We therefore suggest that a transversal filament consists of one or more pairs of SCP1 dimers, each pair being organized in a head-to-head arrangement with the C-termini anchored in the lateral elements and the two N-termini being joined in the central element.

Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7166 - 71
Heterodimer formation and activity in the human enzyme galactose-1-phosphate uridylyltransferase; Elsevier JP et al.; One of the fundamental questions concerning expression and function of dimeric enzymes involves the impact of naturally occurring mutations on subunit assembly and heterodimer activity . This question is of particular interest for the human enzyme galactose-l-phosphate uridylyl-transferase (GALT), impairment of which results in the inherited metabolic disorder galactosemia, because many if not most patients studied to date are compound heterozygotes rather than true molecular homozygotes . Furthermore, the broad range of phenotypic severity observed in these patients raises the possibility that allelic combination, not just allelic constitution, may play some role in determining outcome . In the work described herein, we have selected two distinct naturally occurring null mutations of GALT, Q188R and R333W, and asked the questions (i) what are the impacts of these mutations on subunit assembly, and (ii) if heterodimers do form, are they active? To answer these questions, we have established a yeast system for the coexpression of epitope-tagged alleles of human GALT and investigated both the extent of specific GALT subunit interactions and the activity of defined heterodimer pools . We have found that both homodimers and heterodimers do form involving each of the mutant subunits tested and that both heterodimer pools retain substantial enzymatic activity . These results are significant not only in terms of their implications for furthering our understanding of galactosemia and GALT holoenzyme structure-function relationships but also because the system described may serve as a model for similar studies of other complexes composed of multiple subunits.

Regul Pept, 1996 Jul 5, 63(2-3), 79 - 83
Involvement of beta-endorphin in the modulation of paw inflammatory edema in the rat; Sacerdote P et al.; This study investigates the role of the opiod receptors and of the opioid peptide beta-endorphin in the development of yeast-induced inflammation in the rat paw . Pretreatment with the opioid receptor antagonist naltrexone (10 mg/kg i.p.) exacerbates the paw edema, while morphine pretreatment (5 and 10 mg/kg) reduces it . In addition, the intravenous injection of a specific anti-beta-endorphin antibody aggravates the yeast-induced inflammation . On the contrary, both the kappa-opioid receptor antagonist MR 1452 (2.5, 5 and 10 mg/kg i.p.) and the delta-opioid receptor antagonist ICI 174-864 (2.5, 5 and 10 mg/kg i.p.) do not interfere with the inflammatory process . After intraplantar injection, naltrexone, morphine and the anti-beta-endorphin antibody do not interfere with the yeast-induced inflammatory edema . Our data suggest that beta-endorphin exerts an inhibitory regulation on the inflammatory responses through the activation of mu-opioid receptors probably located on immune cells, rather than in the paw.

Science, 1996 Jul 5, 273(5271), 112 - 5
Evidence for physical and functional association between EMB-5 and LIN-12 in Caenorhabditis elegans; Hubbard EJ et al.; The Caenorhabditis elegans LIN-12 and GLP-1 proteins are members of the LIN-12/Notch family of receptors for intercellular signals that specify cell fate . Evidence presented here suggests that the intracellular domains of LIN-12 and GLP-1 interact with the C . elegans EMB-5 protein and that the emb-5 gene functions in the same pathway as the lin-12 and glp-1 genes . EMB-5 is similar in sequence to a yeast protein that controls chromatin structure . Hence, a direct consequence of LIN-12 or GLP-1 activation may be an alteration of chromatin structure that produces changes in transcriptional activity.

Genes Cells, 1996 Jul, 1(7), 615 - 32
Rab3A small GTP-binding protein in Ca(2+)-dependent exocytosis; Takai Y et al.; There exists a small GTP-binding protein (G protein) superfamily, consisting of more than 50 members, from yeast to mammal . The Rab family belongs to this superfamily and is implicated in intracellular vesicle trafficking . Rab3A small G protein is a member of the Rab3 subfamily which belongs to this Rab family . The regulators and downstream targets of Rab3A have been isolated, and evidence is accumulating that Rab3A and these Rab3A-interacting proteins are involved in Ca(2+)-dependent exocytosis, particularly in neurotransmitter release from nerve terminals.

Anal Chem, 1996 Jul 1, 68(13), 2259 - 63
Determination of nucleic acids by a resonance light-scattering technique with alpha, beta, gamma, delta-tetrakis{4-(trimethylammoniumyl)phenyl}porphine; Huang CZ et al.; The resonance light-scattering technique, using a spectrofluorometer, was first developed as a sensitive instrumental analysis method . At pH 7.48 and ionic strength 0.004, the extent of light-scattering of alpha, beta, gamma, delta-tetrakis{4-(trimethylammoniumyl)phenyl}porphine (TAPP) is enhanced by nucleic acids near 432 nm . There are linear relationships between the enhanced extents of light-scattering and the concentrations of nucleic acids in the range of 1.8 x 10(-7)-10.8 x 10(-7) M for calf thymus and fish sperm DNA and in the range of 1.8 x 10(-7)-1.8 x 10(-6) M for yeast RNA . The limit of determination (3 sigma) is 4.1 x 10(-8) M for calf thymus DNA, 4.6 x 10(-8) M for fish sperm DNA, and 6.7 x 10(-8) M for yeast RNA . Mechanism study indicates that nucleic acids react with the title porphyrin in two modes, depending on the concentrations of nucleic acids . When the molar ratio of nucleic acids to TAPP is smaller than 4:1, the hypochromicity and fluorescence quenching of TAPP by nucleic acids appear, and the enhancement of resonance light-scattering can be observed . When the molar ratio of nucleic acids to TAPP is larger than 4:1, a new fluorescent complex is formed.

Vopr Virusol, 1996 Jul-Aug, 41(4), 170 - 2
{Russian recombinant vaccine against hepatitis B (results of controlled trials)}; Pavlova LI et al.; Publication Types:

	Clinical Trial
	Randomized Controlled Trial

What Is Listeria Monocytogenes?, What Is MIC?, What Is Amino Acid?, What Is Protein?, What Is Molecular Microbiology?, e, Microbes, s, Bacterium, o, Microorganisms, s, Bacteriology, a, Microbe, a, Saccharomyces yeast, c, Candida tropicalis, a, S. cerevisiae, s, Clostridia, e, Bacteriological, r, S. cerevisiae, a, Cryptococci, i, Yeasts, i, Streptococcal, a, Pseudomonas aeruginosa, c, Escherichia coli, c, Cell suspensions, a, S. cerevisiae, r, Bacteroides, o, Antibiotics, o, Enterobacters, e, Microorganism, s, Sepsis, s, Sepsis, a, Bacteriophages, o, Amikacin

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005