Yeasts

Cytogenet Cell Genet, 1996, 72(1), 72 - 7
Regional assignment of EST sequences on human chromosome 13; Hawthorn LA et al.; We have used a panel of somatic cell hybrids carrying structural rearrangements of human chromosome 13 to regionally localise 10 expressed sequence tags (ESTs) from this chromosome . Three of these genes were not present in the somatic cell hybrid PGME, which contains chromosome 13 as its major human component . Using a panel of somatic cell hybrids, two of these genes were localised to chromosome 8 and one to chromosome 16 . Using the PCR primers as published, it was impossible to map one EST; consequently, a YAC containing this gene was isolated, and fluorescence in situ hybridisation was used to map the gene to 13q14 . The distribution of the remaining six chromosome 13-specific ESTs was nonrandom; one mapped to 13q14 and the remaining five to 13q12, with three mapping within the proximal portion and two mapping within the distal portion of this region.

Cytogenet Cell Genet, 1996, 72(1), 63 - 8
A 3.1-Mb YAC contig within the Werner syndrome region, on the short arm of human chromosome 8; Chaffanet M et al.; The locus for Werner syndrome (WRN) has been localized to human chromosome 8p21 --> p12, close to the anonymous marker D8S339 . A 3.1-Mb contig of yeast artificial chromosomes (YACs) was assembled around D8S339 . Results from analyses of somatic cell hybrids, FISH, and physical mapping suggest the following loci order: tel-NEFL-D8S131-D8S339-{D8S540/GSR}-D8S124 -D8S259-D8S87-FGFR1-cen . Close physical linkage between D8S540 and GSR was established within a DNA fragment of 200 kb . These two loci are not more than 400 kb from D8S339 . In addition, D8S339, D8S540, D8S124, and GSR are within 1.1 Mb . These data establish a primary physical map of the Werner syndrome region and identify useful YAC clones for the isolation of new markers and of the corresponding gene.

J Eukaryot Microbiol, 1996 Jan-Feb, 43(1), 61 - 4
In vitro excystation of Spironucleus muris; Koudela B et al.; In vitro excystation of Spironucleus muris cysts, purified by sequential sucrose and Percoll gradients from mouse feces, was studied . Three in vitro excystation procedures, used for Giardia, were assessed to determine the most useful method . Excystation was monitored by light microscopy and subsequently characterized by transmission and scanning electron microscopy . Spironucleus muris excysted routinely at a level greater than 90% when induced in Hanks' balanced salt solution containing sodium bicarbonate at pH 2.0 and transferred to Tyrodes' salt solution as an excystation medium . Similarly, high rates of excystation were recorded after induction of S . muris cysts in 0.1 M potassium phosphate buffer (pH 7.0) with sodium bicarbonate and excystation in trypticase-yeast extract-iron medium (TYI medium) or phosphate-buffered saline . A lower rate and percentage of excystation were observed after induction of S . muris cysts in an aqueous hydrochloric acid solution (pH 2.0) followed by excystation in TYI medium . All excystation methods produced extremely active S . muris trophozoites with normal morphology . Nonexcysting S . muris cysts have a wall composed of an outer fibrous and an inner membranous portion . Following induction, numerous vesicles appeared in the peritrophic space . Excystation began by the cyst wall opening at one pole, and the anterior part of the trophozoite protruding from the cyst wall . The trophozoite emerged progressively from the cyst wall and the empty cyst wall appeared to collapse . Excysted trophozoites exhibited normal morphological features of S . muris trophozoites isolated from the mouse intestine.

Hum Genet, 1996 Jan, 97(1), 60 - 8
Construction of a YAC contig and an STS map spanning 3.6 megabase pairs in Xp22.1; Trump D et al.; We have constructed a 3.6 Mb sequence tagged sites (STS)-based yeast artificial chromosome (YAC) contig, consisting of 58 individual YAC clones, spanning the region PDHA1 and DXS451 on Xp22.1 . In addition to establishing the order of PDHA1, ISPK-1, DXS2504, DXS1528 and the 13 known polymorphic loci as Xpter-PDHA1-DXS443-DXS3424-ISPK-1-DXS12 29-DXS2504-DXS1528-DXS365-DXS7101- DXS1683-DXS1052-DXS274-DXS92-DXS1226-DX S41-DXS989-DXS451-Xcen, we have also developed 35 novel STSs from YAC end clones . These results provide a high density of STS markers (approximately 1 per 70 kb) . Furthermore, a detailed long-range restriction map of the contig has been constructed with rare-cutter enzymes and this has refined and verified the physical distances between markers inferred from YAC sizes and their STS content . The integration of the physical mapping data with previous genetic mapping data and the use of STSs and non-chimeric YAC clones reported here should facilitate the construction of a transcript map of this region and the positional cloning of disease genes in this portion of Xp22.1.

Am J Hum Genet, 1996 Jan, 58(1), 154 - 60
Molecular definition of breakpoints associated with human Xq isochromosomes: implications for mechanisms of formation; Wolff DJ et al.; To test the centromere misdivision model of isochromosome formation, we have defined the breakpoints of cytogenetically monocentric and dicentric Xq isochromosomes (i(Xq)) from Turner syndrome probands, using FISH with cosmids and YACs derived from a contig spanning proximal Xp . Seven different pericentromeric breakpoints were identified, with 10 of 11 of the i(Xq)s containing varying amounts of material from Xp . Only one of the eight cytogenetically monocentric i(Xq)s demonstrated a single alpha-satellite (DXZ1) signal, consistent with classical models involving centromere misdivision . The remaining seven were inconsistent with such a model and had breakpoints that spanned proximal Xp11.21: one was between DXZ1 and the most proximal marker, ZXDA; one occurred between the duplicated genes, ZXDA and ZXDB; two were approximately 2 Mb from DXZ1; two were adjacent to ALAS2 located 3.5 Mb from DXZ1; and the largest had a breakpoint just distal to DXS1013E, indicating the inclusion of 8 Mb of Xp DNA between centromeres . The three cytologically dicentric i(Xq)s had breakpoints distal to DXS423E in Xp11.22 and therefore contained > or = 12 Mb of DNA between centromeres . These data demonstrate that the majority of breakpoints resulting in i(Xq) formation are in band Xp11.2 and not in the centromere itself . Therefore, we hypothesize that the predominant mechanism of i(Xq) formation involves sequences in the proximal short arm that are prone to breakage and reunion events between sister chromatids or homologous X chromosomes.

Am J Hum Genet, 1996 Jan, 58(1), 126 - 32
Fine mapping of the EDA gene: a translocation breakpoint is associated with a CpG island that is transcribed; Srivastava AK et al.; In order to identify the gene for human X-linked anhidrotic ectodermal dysplasia (EDA), a translocation breakpoint in a female with t(X;1)(q13.1;p36.3) and EDA (patient AK) was finely mapped . The EDA region contains five groups of rare-cutter restriction sites that define CpG islands . The two more centromeric of these islands are associated with transcripts of 3.5 kb and 1.8 kb . The third CpG island maps within <1 kb of the translocation breakpoint in patient AK, as indicated by a genomic rearrangement, and approximately 100 kb centromeric from another previously mapped translocation breakpoint (patient AnLy) . Northern analysis with a probe from this CpG island detected an approximately 6-kb mRNA in several fetal tissues tested . An extended YAC contig of 1,200 kb with an average of fivefold coverage was constructed . The two most telomeric CpG islands map 350 kb telomeric of the two translocations . Taken together, the results suggest that the CpG island just proximal of the AK translocation breakpoint lies at the 5' end of a candidate gene for EDA.

Virology, 1996 Jan 1, 215(1), 31 - 9
Mechanism of interferon action . Biochemical and genetic evidence for the intermolecular association of the RNA-dependent protein kinase PKR from human cells; Ortega LG et al.; The interferon-inducible protein kinase (PKR) is activated by an RNA-dependent autophosphorylation . Structure-function studies of the 551 amino acid PKR kinase from human cells have revealed that catalytic-deficient PKR mutants such as PKR(1-551)K296R display a dominant negative behavior when expressed in transfected cells . The potential for PKR to form protein multimers has therefore been examined . Three types of studies, including both genetic and biochemical analyses, demonstrated that PKR from human cells undergoes an intermolecular association that is not dependent upon RNA . First, the intermolecular association of PKR in vitro was demonstrated in the context of an enzyme-substrate interaction . Purified recombinant histidine-tagged PKR(1-551)K296R mutant protein was phosphorylated by purified wild-type PKR; this intermolecular phosphorylation of PKR was dependent on double-stranded RNA . At a fixed RNA concentration, high concentrations of the HIS-PKR(1-551)K296R mutant both impaired the autophosphorylation of wild-type PKR and blocked the trans-phosphorylation of itself . Second, the yeast two-hybrid system was used to probe the intermolecular association of PKR in vivo . Coexpression of the full-length catalytic-deficient phosphotransfer mutant PKR(1-551)K296R as a fusion protein with the Gal4 activation domain and the Gal4 DNA binding domain resulted in the expression of two Gal4-responsive reporter genes, HIS3 and lacZ . The full-length RNA-binding deficient PKR(1-551)K64E/K296R double mutant also interacted with PKR(1-551)K296R sufficiently to activate Gal4-responsive reporter genes; however, other PKR mutants including PKR(1-280)wt and PKR(281-551)K296R as well as p53, RAS, and BCL2 did not . Third, both PKR(1-551)K296R and PKR(1-551)K64E/K296R enhanced the expression of the reovirus S1 gene and S1/S4 chimeric gene in cotransfected COS cells . By contrast, the expression of the reovirus S4 gene was not enhanced by cotransfection with either PKR(1-551)K296R or PKR(1-551)K64E/K296R . These results indicate that PKR interacts with itself in an intermolecular manner both in vivo and in vitro, and that RNA binding is neither necessary nor sufficient for PKR multimerization.

Blood, 1996 Jan 1, 87(1), 324 - 30
Molecular characterization of 12p abnormalities in hematologic malignancies: deletion of KIP1, rearrangement of TEL, and amplification of CCND2; Hoglund M et al.; Twenty patients with hematologic malignancies with 12p abnormalities were investigated by fluorescence in situ hybridization (FISH) using probes mapped to specific regions in 12p . The initial analysis using the YAC 964c10 (D12S736) revealed that all four cases with cytogenetically identified del(12p) had lost one copy of this YAC and that submicroscopic deletions had occurred in 10 of the 16 neoplasms with other 12p abnormalities, ie, translocations, additions, and insertions . The deletions were partially mapped with cosmids localized to subregions of 12p . One copy of the gene for p27kip1 (KIP1), involved in cell cycle entrance, was found to be lost in all cases in which deletions could be detected by other probes and in one case with a translocation as the only detectable change . This implicates KIP1 as a possible tumor suppressor gene affected by del(12p) . Four translocations with no apparent concomitant deletions were detected . All four breakpoints resulted in a split D12S736 signal . In two of these cases, we showed that TEL was disrupted as a result of a t(5;12)(q32-33;p12) and a t(12;22)(p12;q12), respectively . Two lymphoid neoplasm--one non-Hodgkin's lymphoma and one Burkitt's lymphoma--with 12p amplifications were detected . In both cases cyclin D2 (CCND2) was within the amplified region . Thus, cytogenetic abnormalities of 12p in hematologic malignancies result in at least three different molecular changes: deletions of KIP1, amplifications of CCND2, and structural rearrangements of TEL.

Mol Cell Biol, 1996 Jan, 16(1), 37 - 44
p95vav associates with the nuclear protein Ku-70; Romero F et al.; The proto-oncogene vav is expressed solely in hematopoietic cells and plays an important role in cell signaling, although little is known about the proteins involved in these pathways . To gain further information, the Src homology 2 (SH2) and 3 (SH3) domains of Vav were used to screen a lymphoid cell cDNA library by the yeast two-hybrid system . Among the positive clones, we detected a nuclear protein, Ku-70, which is the DNA-binding element of the DNA-dependent protein kinase . In Jurkat and UT7 cells, Vav is partially localized in the nuclei, as judged from immunofluorescence and confocal microscopy studies . By using glutathione S-transferase fusion proteins derived from Ku-70 and coimmunoprecipitation experiments with lysates prepared from human thymocytes and Jurkat and UT7 cells, we show that Vav associates with Ku-70 . The interaction of Vav with Ku-70 requires only the 150-residue carboxy-terminal portion of Ku-70, which binds to the 25 carboxy-terminal residues of the carboxy SH3 domain of Vav . A proline-to-leucine mutation in the carboxy SH3 of Vav that blocks interaction with proline-rich sequences does not modify the binding of Ku-70, which lacks this motif . Therefore, the interaction of Vav with Ku-70 may be a novel form of protein-protein interaction . The potential role of Vav/Ku-70 complexes is discussed.

Gene, 1995 Dec 29, 167(1-2), 209 - 13
Cloning and characterization of a Brassica napus gene encoding a homologue of the B subunit of a heteromeric CCAAT-binding factor; Albani D et al.; The CCAAT motif present in the promoter of several genes is recognized in yeast and animals by a highly specific heteromeric factor (variously called HAP, CBF, CP1 or NF-Y) which is composed of a minimum of three subunits . A plant homologue of the CBF-B/HAP2 subunit is described for the first time in this report . Sequence comparison of the Brassica napus (Bn) CCAAT-binding factor (CBF) B subunit with the homologous yeast and animal proteins revealed that the critical amino-acid domains involved in DNA binding and subunit assembly are also conserved in plants . Interestingly, the Gln-rich regions found in the animal and yeast proteins, which may be involved in transcriptional activation, are absent in the Bn CBF-B subunit . The analysis of various cDNAs and of a genomic clone revealed the presence of alternatively spliced transcripts which could originate from different promoters.

Gene, 1995 Dec 29, 167(1-2), 157 - 61
Cloning and characterization of the POX2 gene in Candida maltosa; Masuda Y et al.; To study the function of acyl-CoA oxidase in an n-alkane-assimilating yeast, Candida maltosa, we isolated the POX2 gene which is a member of the acyl-CoA oxidase gene family . POX2 had a 2172-bp open reading frame (ORF) encoding an approx . 84-kDa polypeptide (724 amino acids (aa)) and was contiguous to POX4, another member of the acyl-CoA oxidase gene family on the same chromosomal DNA in a convergent arrangement . Northern blot analysis revealed that the expression of POX2 was induced in cells grown on oleic acid, n-tetradecanol and n-tetradecane . By using a gene-disruption technique, we constructed strains (termed P2DD and P4DD) in which both alleles of POX2 and POX4 were disrupted . The P2DD strain was normal in assimilation of various hydrophobic carbon sources, such as n-tetradecane, n-tetradecanol and oleic acid . In contrast, the P4DD strain was defective in its ability to grow on such hydrophobic carbon sources.

J Biol Chem, 1995 Dec 29, 270(52), 31338 - 44
Interaction of calreticulin with protein disulfide isomerase; Baksh S et al.; We report here that calreticulin interacts with protein disulfide isomerase (PDI) . The PDI-calreticulin complex can be dissociated by Zn(2+)-iminodiacetate-substituted Sepharose-agarose chromatography, suggesting that these interactions may be Zn2+-dependent . Direct interaction between calreticulin and PDI is also documented by calreticulin affinity chromatography . PDI was the only pancreatic microsomal protein retained on the calreticulum affinity column . Calreticulin and PDI were identified by their NH2-terminal amino acid sequence analysis, mobilities in SDS-polyacrylamide gel electrophoresis, binding of 45Ca2+, and their reactivity with specific antibodies . Using glutathione S-transferase-calreticulin fusion proteins, we show that PDI interacts strongly with the P-domain and only weakly with the N-domain of calreticulin . Expression of calreticulin domains and PDI as fusion proteins with GAL4 in the yeast two-hybrid system revealed that calreticulin interacted with PDI also under normal cellular conditions . Interaction with PDI required only the NH2-terminal region of the N-domain (amino acid residues 1-83) and the P-domain (amino acid residues 150-240) of calreticulin . Importantly, interaction between calreticulin and PDI led to the modulation of their activities . In the presence of PDI, calreticulin does not bind Ca2+ with high affinity . Calreticulin or the N-domain of calreticulin inhibited PDI ability to refold scrambled RNase A.

J Biol Chem, 1995 Dec 29, 270(52), 30853 - 6
The regions of the Fe65 protein homologous to the phosphotyrosine interaction/phosphotyrosine binding domain of Shc bind the intracellular domain of the Alzheimer's amyloid precursor protein; Fiore F et al.; Fe65 is a protein mainly expressed in several districts of the mammalian nervous system . The search of protein sequence data banks revealed that Fe65 contains two phosphotyrosine interaction (PID) or phosphotyrosine binding (PTB) domains, previously identified in the Shc adaptor molecule . The two putative PID/PTB domains of Fe65 were used to construct glutathione S-transferase-Fe65 fusion proteins . Co-precipitation experiments demonstrated that the Fe65 PID/PTB domains interacted with several proteins of apparent molecular mass 135, 115, 105, and 51 kDa . The region of Fe65 containing the PID/PTB domains was used as a bait to screen a human brain cDNA library in yeast by the two-hybrid system . Three different cDNA clones were isolated, two of which contain overlapping segments of the cDNA encoding the COOH terminus of the Alzheimer's beta-amyloid-precursor protein (APP), that represents the short intracellular domain of this membrane protein . The third clone contains a cDNA fragment coding for the COOH terminus of the human counterpart of a mouse beta-amyloid-like precursor protein . The alignment of the three APP encoding cDNA fragments found in the screening suggests that the region of APP involved in the binding is centered on the NPTY sequence, which is analogous to that present in the intracellular domains of the growth factor receptors interacting with the PID/PTB domain of Shc.

Eur J Pharmacol, 1995 Dec 27, 294(1), 71 - 4
Carbamazepine exerts anti-inflammatory effects in the rat; Bianchi M et al.; In a first set of experiments, we evaluated the effects of different doses (5.0, 10, 20 and 40 mg/kg p.o.) of carbamazepine on nociceptive thresholds to thermal and mechanical stimuli, and on paw inflammatory hyperalgesia induced by the injection of brewer's yeast . Moreover, we studied the effect of carbamazepine on paw inflammatory edema by plethysmometry . Carbamazepine did not modify nociceptive latencies, but dose dependently reduced the hyperalgesia and the edema induced by the brewer's yeast injection in the rat hindpaw . In a second set of experiments, we studied the effects induced by the same doses of the drug on subcutaneous carrageenin-induced inflammation . Carbamazepine dose dependently reduced the inflammatory exudate, the prostaglandin E2-like activity in the exudate, and the substance P concentrations in the exudate . Our results demonstrate that carbamazepine is able to inhibit the development of different types of inflammation in the rat.

FEBS Lett, 1995 Dec 27, 377(3), 429 - 33
Human REG family genes are tandemly ordered in a 95-kilobase region of chromosome 2p12; Miyashita H et al.; Reg, first isolated from a rat regenerating islet cDNA library, is expressed in regenerating islet beta-cells . Recently, it has been revealed that Reg and Reg-related genes constitute a multigene family, the Reg family . In human, the four REG family genes, i.e., REG 1 alpha, REG 1 beta, REG-related sequence (RS) and HIP/PAP, have so far been isolated . In this study, we analyzed YAC clones containing the four genes and performed two-color FISH to determine the map order of the genes . The human REG family genes are tandemly ordered in the 95-kbp DNA region of chromosome 2p12 as follows: 2cen-HIP/PAP-RS-REG I alpha-REG I beta-ptel.

Nucleic Acids Res, 1995 Dec 25, 23(24), 5012 - 9
Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells; Smih F et al.; Double-strand breaks (DSBs) are recombinogenic lesions in chromosomal DNA in yeast, Drosophila and Caenorhabditis elegans . Recent studies in mammalian cells utilizing the I-Scel endonuclease have demonstrated that in some immortalized cell lines DSBs in chromosomal DNA are also recombinogenic . We have now tested embryonic stem (ES) cells, a non-transformed mouse cell line frequently used in gene targeting studies . We find that a DSB introduced by I-Scel stimulates gene targeting at a selectable neo locus at least 50-fold . The enhanced level of targeting is achieved by transient expression of the I-Scel endonuclease . In 97% of targeted clones a single base pair polymorphism in the transfected homologous fragment was incorporated into the target locus . Analysis of the targeted locus demonstrated that most of the homologous recombination events were 'two-sided', in contrast to previous studies in 3T3 cells in which 'one-sided' homologous events predominated . Thus ES cells may be more faithful in incorporating homologous fragments into their genome than other cells in culture.

Science, 1995 Dec 22, 270(5244), 1945 - 54
An STS-based map of the human genome; Hudson TJ et al.; A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases . The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci . This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps . The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome . The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized . The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

Oncogene, 1995 Dec 21, 11(12), 2689 - 97
The BTB/POZ domain targets the LAZ3/BCL6 oncoprotein to nuclear dots and mediates homomerisation in vivo; Dhordain P et al.; The LAZ3/BCL6 gene was identified by its disruption in 3q27 translocations associated with diffuse large cell lymphomas . It is predicted to be a transcription factor as it contains six Kruppel-like Zinc finger motifs and a N-terminal BTB/POZ domain, a protein/protein interaction interface that is widely conserved in Metazoans . Using two antisera raised against non overlapping regions of the predicted ORF, we demonstrate that the LAZ3/BCL6 protein appears as a close ca . 79 kDa doublet in B lymphoid cell lines with either a rearranged or a non rearranged LAZ3/BCL6 locus . By immunofluorescence experiments on transiently transfected COS-1 or NIH3T3 cells, we show that the LAZ3/BCL6 protein displays a punctuated nuclear localisation . This appears to rely on LAZ3/BCL6 proper folding and/or activities as it is impaired in a hormone reversible-fashion through fusion of LAZ3/BCL6 to the ligand-binding domain of the oestrogen receptor . Moreover, deletion of its BTB/POZ domain leads to the disappearance of the nuclear dots although the protein remains nuclear . In addition, by using the yeast two-hybrid system, we show that the LAZ3/BCL6 BTB/POZ domain homomerises in vivo . Thus, the LAZ3/BCL6 BTB/POZ domain has the capability to self-interact and target the protein to discrete nuclear substructures.

Oncogene, 1995 Dec 21, 11(12), 2477 - 86
Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression; Jiang H et al.; Cultured human melanoma cells lose proliferative capacity and terminally differentiate after treatment with the combination of recombinant human fibroblast interferon (IFN-beta) and mezerein (MEZ) . Subtraction hybridization of cDNA libraries prepared from actively proliferating human H0-1 melanoma cells from cDNA libraries produced from H0-1 cells treated with IFN-beta + MEZ identifies a novel melanoma differentiation-associated (mda) cDNA, mda-7, that displays elevated expression in differentiation inducer-treated H0-1 cells . mda-7 encodes a novel protein of 206 amino acids with a predicted size of 23.8 kDa . The level of mda-7 mRNA is elevated in actively proliferating normal human melanocytes versus primary and metastatic human melanomas . In the Matrigel-assisted melanoma progression model, mda-7 expression decreases in early vertical growth phase primary human melanoma cells selected for autonomous or enhanced tumor formation in nude mice . Treatment of human melanomas with IFN-beta + MEZ, and to a lesser extent with MEZ, results in growth suppression and induced or enhanced mda-7 expression . Immunoprecipitation analyses using peptide-derived rabbit polyclonal antibodies detect increases in mda-7 protein, and a higher molecular weight protein of approximately 90 to 100 kDa, in MEZ and IFN-beta + MEZ treated H0-1 cells . mda-7 is a highly conserved gene with an homologous sequence in the genome of yeast . Transfection of mda-7 expression constructs into H0-1 and C8161 human melanoma cells reduces growth and inhibits colony formation . These results confirm that mda-7 has antiproliferative properties in human melanoma cells and in this context may contribute to terminal cell differentiation . The mda-7 gene may also function as a negative regulator of melanoma progression.

Nature, 1995 Dec 21-28, 378(6559), 789 - 92
Identification of the breast cancer susceptibility gene BRCA2; Wooster R et al.; In Western Europe and the United States approximately 1 in 12 women develop breast cancer . A small proportion of breast cancer cases, in particular those arising at a young age, are attributable to a highly penetrant, autosomal dominant predisposition to the disease . The breast cancer susceptibility gene, BRCA2, was recently localized to chromosome 13q12-q13 . Here we report the identification of a gene in which we have detected six different germline mutations in breast cancer families that are likely to be due to BRCA2 . Each mutation causes serious disruption to the open reading frame of the transcriptional unit . The results indicate that this is the BRCA2 gene.

Biochemistry, 1995 Dec 19, 34(50), 16235 - 9
A noncanonical tertiary conformation of a human mitochondrial transfer RNA; Leehey MA et al.; Transfer RNAs possess highly conserved secondary structures, and crystallographic studies suggest a common, L-shaped tertiary conformation in which the anticodon and acceptor stems are disposed at approximately right angles to one another . However, many animal mitochondrial tRNAs possess unusual secondary structures, and little is known regarding their tertiary conformations, in particular, the relative orientations of their acceptor and anticodon stems . To address this issue, we have constructed heteroduplex RNA molecules corresponding to human mitochondrial and cytoplasmic lysyl tRNAs in which the acceptor and anticodon stems of each tRNA have been extended by approximately 70 base pairs . The rotational decay times of the two "extended" tRNA(Lys) species were compared to the decay times of a linear RNA control and to an extended yeast cytoplasmic tRNA(Phe) species whose interstem angle had been reported previously . Whereas the apparent interstem angle of the human cytoplasmic tRNA(Lys) species is essentially identical to that of the yeast tRNA(Phe) heteroduplex, with both conforming to the canonical L-shape, the angle for the mitochondrial tRNA(Lys) construct is much larger (approximately 140 degrees) . Thus, the universal L-shape may not be applicable to noncanonical mitochondrial tRNAs, a finding of significance for both tRNA evolution and mitochondrial disease.

Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12456 - 60
Multiple genetic loci within 11p15 defined by Beckwith-Wiedemann syndrome rearrangement breakpoints and subchromosomal transferable fragments; Hoovers JM et al.; Beckwith-Wiedemann syndrome (BWS) involves fetal overgrowth and predisposition to a wide variety of embryonal tumors of childhood . We have previously found that BWS is genetically linked to 11p15 and that this same band shows loss of heterozygosity in the types of tumors to which children with BWS are susceptible . However, 11p15 contains > 20 megabases, and therefore, the BWS and tumor suppressor genes could be distinct . To determine the precise physical relationship between these loci, we isolated yeast artificial chromosomes, and cosmid libraries from them, within the region of loss of heterozygosity in embryonal tumors . Five germ-line balanced chromosomal rearrangement breakpoint sites from BWS patients, as well as a balanced chromosomal translocation breakpoint from a rhabdoid tumor, were isolated within a 295- to 320-kb cluster defined by a complete cosmid contig crossing these breakpoints . This breakpoint cluster terminated approximately 100 kb centromeric to the imprinted gene IGF2 and 100 kb telomeric to p57KIP2, an inhibitor of cyclin-dependent kinases, and was located within subchromosomal transferable fragments that suppressed the growth of embryonal tumor cells in genetic complementation experiments . We have identified 11 transcribed sequences in this BWS/tumor suppressor coincident region, one of which corresponded to p57KIP2 . However, three additional BWS breakpoints were > 4 megabases centromeric to the other five breakpoints and were excluded from the tumor suppressor region defined by subchromosomal transferable fragments . Thus, multiple genetic loci define BWS and tumor suppression on 11p15.

Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12180 - 4
Identification and expression analysis of a potential familial Alzheimer disease gene on chromosome 1 related to AD3; Li J et al.; The inheritance of much early-onset Alzheimer disease (AD) has been linked to a dominant-acting locus on chromosome 14 . Recently, the gene likely responsible for this genetic linkage has been identified and termed AD3 . Five mutations have been found in AD3 that segregate with the disease phenotype in seven AD families and are not present in unaffected individuals . Here we report the existence of a gene encoding a seven transmembrane domain protein very similar to that encoded by AD3 in structure and sequence . This gene is located on chromosome 1, is expressed in a variety of tissues, including brain, and is predicted to harbor mutations causing nonchromosome 14 familial AD . The presence of several S/TPXX DNA binding motifs in both the AD3 protein and the AD3-like protein /AD4 protein suggests a possible role in intracellular signaling and gene expression or in linking chromatin to the nuclear membrane . Ways in which mutations in either gene could lead to AD are discussed.

Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12160 - 4
Domains with transcriptional regulatory activity within the ALL1 and AF4 proteins involved in acute leukemia; Prasad R et al.; The ALLI gene, located at chromosome band 11q23, is involved in acute leukemia through a series of chromosome translocations and fusion to a variety of genes, most frequently to A4 and AF9 . The fused genes encode chimeric proteins proteins . Because the Drosophila homologue of ALL1, trithorax, is a positive regulator of homeotic genes and acts at the level of transcription, it is conceivable that alterations in ALL1 transcriptional activity may underlie its action in malignant transformation . To begin studying this, we examined the All1, AF4, AF9, and AF17 proteins for the presence of potential transcriptional regulatory domains . This was done by fusing regions of the proteins to the yeast GAL4 DNA binding domain and assaying their effect on transcription of a reporter gene . A domain of 55 residues positioned at amino acids 2829-2883 of ALL1 was identified as a very strong activator . Further analysis of this domain by in vitro mutagenesis pointed to a core of hydrophobic and acidic residues as critical for the activity . An ALL1 domain that repressed transcription of the reporter gene coincided with the sequence homologous to a segment of DNA methyltransferase . An AF4 polypeptide containing residues 480-560 showed strong activation potential . The C-terminal segment of AF9 spanning amino acids 478-568 transactivated transcription of the reporter gene in HeLa but not in NIH 3T3 cells . These results suggest that ALL1, AF4, and probably AF9 interact with the transcriptional machinery of the cell.

Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12036 - 40
Seed and vascular expression of a high-affinity transporter for cationic amino acids in Arabidopsis; Frommer WB et al.; In most plants amino acids represent the major transport form for organic nitrogen . A sensitive selection system in yeast mutants has allowed identification of a previously unidentified amino acid transporter in Arabidopsis . AAT1 encodes a hydrophobic membrane protein with 14 membrane-spanning regions and shares homologies with the ecotropic murine leukemia virus receptor, a bifunctional protein serving also as a cationic amino acid transporter in mammals . When expressed in yeast, AAT1 mediates high-affinity transport of basic amino acids, but to a lower extent also recognizes acidic and neutral amino acids . AAT1-mediated histidine transport is sensitive to protonophores and occurs against a concentration gradient, indicating that AAT1 may function as a proton symporter . AAT1 is specifically expressed in major veins of leaves and roots and in various floral tissues--i.e., and developing seeds.

FEBS Lett, 1995 Dec 18, 377(2), 243 - 8
A novel partner for the GTP-bound forms of rho and rac; Madaule P et al.; Using the yeast two hybrid system and overlay assays we identified a putative rholrac effector, citron, which interacts with the GTP-bound forms of rho and rac1, but not with cdc42 . Extensive homologies to known proteins were not observed . This 183 kDa protein contains a C6H2 zinc finger, a PH domain, and a long coiled-coil forming region including 4 leucine zippers and the rholrac binding site . We recently identified three others putative rho effectors characterized by a common rho binding motif . Citron does not share this motif and displays a distinctive protein organization, thus defining a separate class of rho partners.

Eur J Biochem, 1995 Dec 15, 234(3), 723 - 31
Identification of spinach farnesyl protein transferase . Dithiothreitol as an acceptor in vitro; Parmryd I et al.; Spinach seedlings were found to contain farnesyl protein transferase . The enzyme is activated by Zn2+, but not by Mg2+ . The pH optimum is approximately 7.0 and maximal activity is obtained at 40-45 degrees C . The apparent Km for the farnesyl diphosphate substrate is 7 microM . Western blotting of soluble proteins with an antiserum raised against mammalian farnesyl protein transferase demonstrated a specific cross-reactivity with the spinach enzyme . The antiserum preferentially recognises the beta-subunit of the heterodimeric farnesyl protein transferase, and the corresponding spinach polypeptide has a molecular mass of 42 kDa on SDS/PAGE . The enzyme can employ dithiothreitol as an acceptor for the farnesyl moiety and catalyses the formation of a thioether linkage between these substrates . On the basis of this discovery, a new method was developed utilising the hydrophobicity of the reaction product, and its interaction with poly(propylene) . During in vivo labelling, the plants took up dithiothreitol, which inhibited the incorporation of {3H}mevalonate metabolites into proteins, indicating that dithiothreitol might be isoprenylated in vivo as well as in vitro . However, isoprenylation of some proteins remains unaffected by dithiothreitol suggesting the existence of different isoprenylation mechanisms . Thus, it is demonstrated that plants possess farnesyl protein transferase, which resembles its mammalian and yeast homologues.

Toxicology, 1995 Dec 15, 104(1-3), 1 - 8
Substrates of human hepatic cytochrome P450 3A4; Li AP et al.; Cytochrome P450 isozyme 3A4 (CYP3A4) is a major isozyme in the human liver and is known to metabolize a larger variety of xenobiotics and endogenous biochemicals . The identities of CYP3A4 substrates are summarized here . A total of 32 chemicals belonging to different structural classes have been evaluated and found to be substrates for CYP3A4 . The metabolic pathways for these substrates include N-oxidation, C-oxidation, N-dealkylation, O-dealkylation, nitro-reduction, dehydration, and C-hydroxylation . While the major experimental system used to elucidate the role of CYP3A4 in the metabolic transformation of these substrates is the human liver microsome system, cultured human hepatocytes and yeast/cultured cells genetically engineered to express CYP3A4 are also employed by the different investigators . The common approaches to identify the role of CYP3A4 are also summarized, which include correlation of metabolic activity of the substrates studied with those for known CYP3A4-catalyzed substrates, correlation of activity with CYP3A4 content, inhibition of activity with CYP3A4 specific antibodies, inhibition of activity with known CYP3A4 substrates and inhibitors, induction of activity with CYP3A4 inducers and demonstration of activity with purified CYP3A4 enzyme.

Genes Dev, 1995 Dec 15, 9(24), 3083 - 96
Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4; Bungert J et al.; Proper tissue- and developmental stage-specific transcriptional control over the five genes of the human beta-globin locus is elicited in part by the locus control region (LCR), but the molecular mechanisms that dictate this determined pattern of gene expression during human development are still controversial . By use of homologous recombination in yeast to generate mutations in the LCR within a yeast artificial chromosome (YAC) bearing the entire human beta-globin gene locus, followed by injection of each of the mutated YACs into murine ova, we addressed the function of LCR hypersensitive site (HS) elements 3 and 4 in human beta-globin gene switching . The experiments revealed a number of unexpected properties that are directly attributable to LCR function . First, deletion of either HS3 or HS4 core elements from an otherwise intact YAC results in catastrophic disruption of globin gene expression at all erythroid developmental stages, despite the presence of all other HS elements in the YAC transgenes . If HS3 is used to replace HS4, gene expression is normal at all developmental stages . Conversely, insertion of the HS4 element in place of HS3 results in significant expression changes at every developmental stage, indicating that individual LCR HS elements play distinct roles in stage-specific beta-type globin gene activation . Although the HS4 duplication leads to alteration in the levels of epsilon- and gamma-globin mRNAs during embryonic erythropoiesis, total beta-type globin mRNA synthesis is balanced, thereby leading to the conclusion that all of the human beta-locus genes are competitively regulated . In summary, the human beta-globin HS elements appear to form a single, synergistic functional entity called the LCR, and HS3 and HS4 appear to be individually indispensable to the integrity of this macromolecular complex.

Genes Dev, 1995 Dec 15, 9(24), 3051 - 66
A proline-rich TGF-beta-responsive transcriptional activator interacts with histone H3; Alevizopoulos A et al.; The molecular mechanisms involved in the regulation of gene expression by transforming growth factor-beta (TGF-beta) have been analyzed . We show that TGF-beta specifically induces the activity of the proline-rich trans-activation domain of CTF-1, a member of the CTF/NF-I family of transcription factors . A TGF-beta-responsive domain (TRD) in the proline-rich transcriptional activation sequence of CTF-1 was shown to mediate TGF-beta induction in NIH-3T3 cells . Mutagenesis studies indicated that this domain is not the primary target of regulatory phosphorylations, suggesting that the growth factor may regulate a CTF-1-interacting protein . A two-hybrid screening assay identified a nucleosome component, histone H3, as a specific CTF-1-interacting protein in yeast . Furthermore, the CTF-1 trans-activation domain was shown to interact with histone H3 in both transiently and stably transfected mammalian cells . This interaction requires the TRD, and it appears to be upregulated by TGF-beta in vivo . Moreover, point mutations in the TRD that inhibit TGF-beta induction also reduce interaction with histone H3 . In vitro, the trans-activation domain of CTF-1 specifically contacts histone H3 and oligomers of histones H3 and H4, and full-length CTF-1 was shown to alter the interaction of reconstituted nucleosomal cores with DNA . Thus, the growth factor-regulated trans-activation domain of CTF-1 can interact with chromatin components through histone H3 . These findings suggest that such interactions may regulate chromatin dynamics in response to growth factor signaling.

J Biol Chem, 1995 Dec 15, 270(50), 29628 - 31
Interaction of the transforming growth factor-beta type I receptor with farnesyl-protein transferase-alpha; Kawabata M et al.; Transforming growth factor-beta 1 (TGF-beta 1) is the prototype of a large family of molecules that regulate a variety of biological processes . The type I (T beta R-I) and type II (T beta R-II) receptors for TGF-beta 1 are transmembrane serine/threonine kinases, forming a heteromeric signaling complex . Recent studies have shown that T beta R-II is a constitutively active kinase and phosphorylates T beta R-I upon ligand binding, suggesting that T beta R-I is the effector subunit of the receptor complex, which transduces signals to intracellular targets . This model has been further confirmed by the identification of constitutively active T beta R-I that mediates TGF-beta 1-specific cellular responses in the absence of ligand and T beta R-II . To investigate signaling by TGF-beta 1, we have sought to isolate proteins that interact with the cytoplasmic region of T beta R-I . One of the proteins identified was the alpha subunit of farnesyl-protein transferase (FT alpha) that modifies a series of peptides including Ras . T beta R-I specifically interacts with FT alpha in the yeast two-hybrid system . Glutathione S-transferase-T beta R-I fusion proteins bind FT alpha translated in vitro . T beta R-I also phosphorylates FT alpha . We further show that the constitutively active T beta R-I interacted with FT alpha very strongly whereas an inactive form of T beta R-I did not . These results suggest that FT alpha may be one of the substrates of the activated T beta R-I kinase.

Cell, 1995 Dec 15, 83(6), 925 - 35
The tomato gene Pti1 encodes a serine/threonine kinase that is phosphorylated by Pto and is involved in the hypersensitive response; Zhou J et al.; The Pto gene encodes a serine/threonine kinase that confers resistance to bacterial speck disease in tomato . Using the yeast two-hybrid system, we identified a second serine/threonine kinase, Pto-interacting 1 (Pti1), that physically interacts with Pto . Cross-phosphorylation assays revealed that Pto specifically phosphorylates Pti1 and that Pti1 does not phosphorylate Pto . Fen, another serine/threonine kinase from tomato that is closely related to Pto, was unable to phosphorylate Pti1 and was not phosphorylated by Pti1 . Expression of a Pti1 transgene in tobacco plants enhanced the hypersensitive response to a P . syringae pv . tabaci strain carrying the avirulence gene avrPto . These findings indicate that Pti1 is involved in a Pto-mediated signaling pathway, probably by acting as a component downstream of Pto in a phosphorylation cascade.

Cell, 1995 Dec 15, 83(6), 1001 - 9
p25rum1 orders S phase and mitosis by acting as an inhibitor of the p34cdc2 mitotic kinase; Correa-Bordes J et al.; p25rum1 from the fission yeast S . pombe is shown to act as a specific in vitro inhibitor of the p34cdc2/p56cdc13 mitotic kinase . It is also shown that early G1 cells contain p25rum1, which associates with and inhibits the mitotic kinase, and maintains p56cdc13 mitotic B cyclin at a low level, ensuring that these cells do not undergo a premature lethal entry into mitosis . A high level of p25rum1 in G2 cells inhibits the p34cdc2/p56cdc13 kinase that removes the block preventing a further S phase and leads to repeated rounds of DNA replication . Thus, the cyclin-dependent kinase inhibitor p25rum1, acting on the p34cdc2 mitotic kinase, plays an important role in ensuring the correct sequence of S phase and mitosis during the cell cycle.

Gene, 1995 Dec 12, 166(2), 337 - 8
Assignment of the E4TF1-60 gene to human chromosome 21q21.2-q21.3; Goto M et al.; The gene encoding human transcription factor E4TF1-60 was previously mapped to chromosome 21q21 . We analyzed the localization of the E4TF1-60 gene in more detail by genomic Southern hybridization and determined the sequence of the exons and the regions surrounding the intron boundaries . We report here that E4TF1-60 locates in the long arm of chromosome 21 at q21.2-q21.3 and contains a total of ten exons.

Genomics, 1995 Dec 10, 30(3), 514 - 20
A 11 Mb YAC-based contig spanning the familial juvenile nephronophthisis region (NPH1) located on chromosome 2q; Konrad M et al.; A gene (NPH1) responsible for approximately 90% of the purely renal form of familial juvenile nephronophthisis, a progressive tubulo-interstitial kidney disorder, maps to human chromosome 2 . We report the construction of a YAC-based contig spanning the critical NPH1 region and the flanking genetic markers . This physical map was integrated with a refined genetic map that restricted the NPH1 interval to about 2 cM; this interval corresponds to a maximum physical distance of 3.5 Mb . The entire contig covers 9 cM between the loci D2S135 and D2S121 . The maximum physical distance between these two markers is approximately 11.3 Mb . Forty-five sequence-tagged sites, including six genes, have been located within this contig . PAX8, a member of the human paired box gene family, that is expressed in the developing kidney, was assigned outside the restricted NPH1 critical region and cannot therefore be regarded as a candidate gene . This set of overlapping clones represents a useful resource for further targeted development of genetic markers and for the characterization of candidate genes responsible for juvenile nephronophthisis.

Genomics, 1995 Dec 10, 30(3), 486 - 92
A precise meiotic map in the class I region of the human major histocompatibility complex; Bouissou C et al.; Human families in which recombinant meiotic event(s) are known to have occurred are powerful tools with which to analyze more precisely the structures of defined genomic regions, especially unstable areas . Such families allow the determination of the haplotypes of each member and, taking into account the recombinant event, it is possible to localize very precisely the point of crossover . Using families in which crossovers between the genes HLA-A and -B have occurred, we have constructed a meiotic map localizing the meiotic breakpoint events with respect to both anonymous markers and the principal genes of the region . Such mapping, which depends on the direct analysis of genomic DNA, is essential for fine structural analysis and is a powerful means of verification of the order and the localization of markers: physical mapping alone, using yeast artificial chromosomes, presents some uncertainties due to the numerous chimeras and inversions that can be produced . The establishment of this map will allow us to determine efficiently the precise location for new markers already localized to the map region . Three microsatellites (D6S265, D6S276, and D6S306), localized in the HLA region by linkage analysis, have been precisely located with respect to the points of recombination in the class I region . The sites of meiotic recombination in the MHC class I region seem to be not randomly distributed but in the majority of cases occurred between HLA-C and the microsatellite D6S265 . This study also shows two cases of abnormal segregation of alleles . We discuss how these mutations correspond to a spontaneous mutation event at the somatic or germinal level.

Genomics, 1995 Dec 10, 30(3), 439 - 44
The Rab protein family: genetic mapping of six Rab genes in the mouse; Barbosa MD et al.; Rab proteins constitute a family of GTP-binding proteins that are located in distinct intracellular compartments and play a role in the regulation of vesicular trafficking . Yeast mutations in Rab gene homologs cause defects in vesicular transport similar to those observed in beige (bg) mice . To investigate Rab genes as candidates for mouse mutations characterized by defects in vesicular trafficking, we utilized an inter-subspecific backcross {C57BL/6J-bgJ x (C57BL/6J-bgJ x CAST/Ei)F1} segregating for the bg locus . Restriction fragment length polymorphisms (RFLPs) were obtained through Southern hybridization of F1 and C57BL/6J chromosomal DNA with the coding sequences of Rab genes . These RFLPs and 12 polymorphic microsatellites were used to determine the segregation of the Rab genes in 93 backcross mice . Rab4a, Rab4b, Rab7, Rab10, Rab22, and Rab24 were localized on mouse chromosomes 8, 7, 9, 12, 2, and 13, respectively . Although the results exclude these loci as candidates for bg, they demonstrate a wide dispersion of Rab genes throughout the mouse genome and reveal that Rab4b and Rab24 are possible candidates for the mouse mutations reduced pigmentation (rp) and purkinje cell degeneration (pcd), respectively.

Science, 1995 Dec 8, 270(5242), 1660 - 3
Sodium-driven potassium uptake by the plant potassium transporter HKT1 and mutations conferring salt tolerance; Rubio F et al.; Sodium (Na+) at high millimolar concentrations in soils is toxic to most higher plants and severely reduces agricultural production worldwide . However, the molecular mechanisms for plant Na+ uptake remain unknown . Here, the wheat root high-affinity potassium (K+) uptake transporter HKT1 was shown to function as a high-affinity K(+)-Na+ cotransporter . High-affinity K+ uptake was activated by micromolar Na+ concentrations; moreover, high-affinity Na+ uptake was activated by K+ (half-activation constant, 2.8 microM K+) . However, at physiologically detrimental concentrations of Na+, K+ accumulation mediated by HKT1 was blocked and low-affinity Na+ uptake occurred (Michaelis constant, approximately 16 mM Na+), which correlated to Na+ toxicity in plants . Point mutations in the sixth putative transmembrane domain of HKT1 that increase Na+ tolerance were isolated with the use of yeast as a screening system . Na+ uptake and Na+ inhibition of K+ accumulation indicate a possible role for HKT1 in physiological Na+ toxicity in plants.

Science, 1995 Dec 8, 270(5242), 1601 - 7
Telomeres: beginning to understand the end; Zakian VA; Telomeres are the protein-DNA structures at the ends of eukaryotic chromosomes . In yeast, and probably most other eukaryotes, telomeres are essential . They allow the cell to distinguish intact from broken chromosomes, protect chromosomes from degradation, and are substrates for novel replication mechanisms . Telomeres are usually replicated by telomerase, a telomere-specific reverse transcriptase, although telomerase-independent mechanisms of telomere maintenance exist . Telomere replication is both cell cycle- and developmentally regulated, and its control is likely to be complex . Because telomere loss causes the kinds of chromosomal changes associated with cancer and aging, an understanding of telomere biology has medical relevance.

Oncogene, 1995 Dec 7, 11(11), 2317 - 29
Structural requirements for the efficient regulation of the Src protein tyrosine kinase by Csk; Koegl M et al.; Protein tyrosine kinases of the Src family are negatively regulated by phosphorylation in the C-terminal tail of the molecule . A different protein tyrosine kinase, Csk, is largely responsible for this regulation . The phosphorylated tail of c-Src engages with the SH2 domain in a conformation that is associated with low kinase activity and which involves stabilization by the SH3 domain . Inducible expression of c-Src in fission yeast is lethal unless Csk is coexpressed . Using this assay we present evidence that Src regulation by C-terminal phosphorylation does not require the myristylation signal or the unique domain at the N-terminus of the Src protein . Mutagenesis of the SH3 and SH2 domains of Csk show that neither are necessary in yeast or in vitro for efficient regulation of Src . Mutation of Tyr416 of Src, a site of autophosphorylation common to most protein tyrosine kinases, abolished the ability of Src to arrest growth of phosphorylate endogenous proteins . Tyr416 had the same effect on a shorter form of Src consisting of the kinase domain only, indicating that the mutation affects a property intrinsic to the catalytic domain . The residual activity of full-length Src mutated at Tyr416 is efficiently repressed by Csk action, suggesting that regulation by C-terminal phosphorylation does not act by preventing phosphorylation at Tyr416.

Nature, 1995 Dec 7, 378(6557), 626 - 9
A phosphate transporter from the mycorrhizal fungus Glomus versiforme; Harrison MJ et al.; Vesicular-arbuscular (VA) mycorrhizal fungi form symbiotic associations with the roots of most terrestrial plants, including many agriculturally important crop species . The fungi colonize the cortex of the root to obtain carbon from their plant host, while assisting the plant with the uptake of phosphate and other mineral nutrients from the soil . This association is beneficial to the plant, because phosphate is essential for plant growth and development, especially during growth under nutrient-limiting conditions . Molecular genetic studies of these fungi and their interaction with plants have been limited owing to the obligate symbiotic nature of the VA fungi, so the molecular mechanisms underlying fungal-mediated uptake and translocation of phosphate from the soil to the plant remain unknown . Here we begin to investigate this process by identifying a complementary DNA that encodes a transmembrane phosphate transporter (GvPT) from Glomus versiforme, a VA mycorrhizal fungus . The function of the protein encoded by GvPT was confirmed by complementation of a yeast phosphate transport mutant . Expression of GvPT was localized to the external hyphae of G . versiforme during mycorrhizal associations, these being the initial site of phosphate uptake from the soil.

Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11894 - 8
A Fas-associated protein factor, FAF1, potentiates Fas-mediated apoptosis; Chu K et al.; Fas, a member of the tumor necrosis factor receptor family, can induce apoptosis when activated by Fas ligand binding or anti-Fas antibody crosslinking . Genetic studies have shown that a defect in Fas-mediated apoptosis resulted in abnormal development and function of the immune system in mice . A point mutation in the cytoplasmic domain of Fas (a single base change from T to A at base 786), replacing isoleucine with asparagine, abolishes the signal transducing property of Fas . Mice homozygous for this mutant allele (lprcg/lprcg mice) develop lymphadenopathy and a lupus-like autoimmune disease . Little is known about the mechanism of signal transduction in Fas-mediated apoptosis . In this study, we used the two-hybrid screen in yeast to isolate a Fas-associated protein factor, FAF1, which specifically interacts with the cytoplasmic domain of wild-type Fas but not the lprcg-mutated Fas protein . This interaction occurs not only in yeast but also in mammalian cells . When transiently expressed in L cells, FAF1 potentiated Fas-induced apoptosis . A search of available DNA and protein sequence data banks did not reveal significant homology between FAF1 and known proteins . Therefore, FAF1 is an unusual protein that binds to the wild type but not the inactive point mutant of Fas . FAF1 potentiates Fas-induced cell killing and is a candidate signal transducing molecule in the regulation of apoptosis.

Biochemistry, 1995 Dec 5, 34(48), 15689 - 99
Increase of the P1 Lys/Leu substrate preference of carboxypeptidase Y by rational design based on known primary and tertiary structures of serine carboxypeptidases; Olesen K et al.; The P1 substrate preference of serine carboxypeptidases, as expressed by the Lys/Leu ratio, differs by up to 10(5)-fold . Predictions of the major determinants of this preference are made by correlating primary and tertiary structures to substrate preferences . In carboxypeptidase Y from yeast it is predicted that Trp312 constitutes such a determinant, reducing the P1 Lys/Leu substrate preference of this enzyme . The predictions are tested by the construction and kinetic characterization of ten mutant enzymes of carboxypeptidase Y . All of these enzymes exhibit changes in their P1 substrate preference . Generally, small decreases in activity (kcat/Km) are observed with substrates containing uncharged P1 side chains . With substrates containing acidic P1 side chains, i.e., FA-Glu-Ala-OH, the activity generally increases slightly, 7-fold in the case of W312K . The most dramatic effects of the Trp312 substitutions are observed with substrates containing basic P1 side chains, i.e., kcat/Km for the hydrolysis of Fa-Lys-Ala-OH with W312E has increased 1150-fold, exclusively as a result of increased kcat values . Similar results have previously been obtained by mutational substitution at position 178 of carboxypeptidase Y . The construction and kinetic characterization of position 178 + 312 double mutants demonstrate that the kinetic effects of substitutions at these two positions are not additive . The P1 Lys/Leu substrate preference of one double mutant, L178D + W312D, has changed 380,000-fold as compared to the wild type enzyme, and the overall P1 substrate preference of this enzyme closely resembles that of carboxypeptidase WII from wheat.

J Autoimmun, 1995 Dec, 8(6), 859 - 74
A family of hsp60-related proteins in pancreatic beta cells of non-obese diabetic (NOD) mice; Brudzynski K et al.; Eukaryotic hsp60s are plastid-specific molecular chaperones implicated in the pathogenesis of many inflammatory and autoimmune diseases . We have used immunoelectron microscopy, immunoblotting and subcellular fractionation of islet cells to determine whether analogous proteins with related function are expressed in other cellular structures and whether such hsp60-related proteins could serve as antigenic targets in autoimmune diabetes . Using a panel of monoclonal and polyclonal antibodies to human and yeast hsp60s and immunoelectron microscopy, the hsp60 antibody cross-reactive proteins were detected in secretory granules, mitochondria, synaptic-like microvesicles and microtubules of mouse pancreatic beta cells . The expression of microtubule-associated hsp60 was induced by an infiltration of islets by mononuclear cells . This novel inducible-form of hsp60-related protein was recognized as an antigen by sera from diabetic mice . Subcellular fractionation of islets indicated that the molecular size of hsp60-related proteins included 66, 62, 58, 55, 52 and 38 kDa . These results demonstrate that the pancreatic beta cells express a family of hsp60-related proteins, with members differentially expressed in distinct cellular compartments . These proteins bearing hsp60 epitopes were antigenic targets for autoimmune responses in diabetic NOD mice.

Clin Exp Allergy, 1995 Dec, 25(12), 1235 - 45
Influence of culture period on the allergenic composition of Pityrosporum orbiculare extracts; Zargari A et al.; BACKGROUND: Previous characterization studies of Pityrosporum orbiculare allergens have led to contradictory results . In immunoblotting studies a range of IgE-binding proteins of 10-100 kDa have been identified . In another study, however, the IgE-binding structures were claimed to be associated with high-molecular-weight polysaccharides or glycoproteins, presumably mannans or mannoproteins . OBJECTIVE: In the present study the reasons for these discrepancies were investigated . METHODS: P . orbiculare preparations were compared in IgE ELISA and IgE-inhibition ELISA, as well as in immunoblotting with sera from atopic dermatitis patients . RESULTS: It was inferred that variations in the period of in vitro culture of P . orbiculare constituted the most important factor determining the different compositions of the resulting yeast cell extracts . After 2 days of culture a wide range of allergenic proteins was present but upon more prolonged culture (> 4 days) most proteins of 10-100 kDa were lost . Accordingly, the protein concentration of the extracts gradually declined from 40% to 25% between days 4 and 15 of culture . On the other hand, the carbohydrate content remained fairly constant (approximately 30%) . Using inhibition ELISA it was demonstrated that the high-molecular-weight glycoproteins or polysaccharides presumably involved in most of the IgE-binding capacity in extracts from old cultures, were also present in comparable concentrations in all extracts tested, even after culture for only 2 or 4 days . CONCLUSION: Preparations obtained from the exponential phase of yeast cultures (2-4 days old), should preferably be used in studies of the IgE response to P . orbiculare.

Comput Appl Biosci, 1995 Dec, 11(6), 657 - 65
A probabilistic algorithm for interactive huge genome comparison; Courtois PR et al.; We designed a new probabilistic algorithm, named PAGEC (probabilistic algorithm for genome comparison), which allowed a highly interactive study of long genomic strings . The comparison between two nucleic acid sequences is based on the creation of multiple index tables, which drastically reduces processing time for huge genomes, e.g . 13 min for a 4 Mb/4 Mb comparison . PAGEC lowered the need for memory when compared with other types of algorithm and took into account the low resolution of the final representation (paper or computer screen) . Considering that standard printers permit a 300 d.p.i . resolution, the loss of computed information due to the probabilistic conception of the algorithm was not usually noticeable in the present study, mainly due to increased genomic sizes . Refinement was possible through an interactive zooming system, which enabled the visualization of the lexical base sequences of a considered part of both of the studied genomes . Biological examples of computation based on yeast and animal nucleic acid sequences presented in this paper reveal the flexibility of the PAGEC program, which is a valuable tool for genetic studies as it offers a solution to an important problem that will become even more important as time passes.

Comput Appl Biosci, 1995 Dec, 11(6), 645 - 55
SAM: a system for iteratively building marker maps; Soderlund C et al.; SAM (system for assembling markers) is a system which supports man-machine problem solving for iteratively ordering a set of markers . SAM aids the user in partially ordering a set of markers based on incomplete and uncertain data . As data is added and modified, SAM aids the user in updating the previously assembled maps . The input is a file of clones and for each clone, a list of the markers contained within it . The objective is to order the set of markers such that the markers contained in each clone are consecutive . The user directs the map building by selecting functions to assemble a region of markers, order the clones to fit the order of the markers and position new markers within an ordered set of markers . The user can edit the input data, edit the assembled map and add clones to the map based on their marker content . The results are displayed graphically and can be saved in a solution file . Based on the partial map, the user designs new experiments or edits the existing data to fill gaps and resolve ambiguities . When a previously assembled map is loaded into SAM, it is automatically updated with the new or altered data . SAM treats all markers as points, but has special features for multiple copy and long markers so that they can be used in the map building process . This system has supported the building of a YAC map of human chromosome 22 at the Sanger Centre, where use of Alu-PCR product markers is a major component in determining clone overlap and where we have an on-going effort to accumulate data from various sources . SAM is also being used at various other laboratories.

Genome Res, 1995 Dec, 5(5), 453 - 63
Assessment of a mutation in the H5 domain of Girk2 as a candidate for the weaver mutation; Mjaatvedt AE et al.; A mutation in the GIRK2 inwardly rectifying K+ channel was mapped recently to the region of mouse chromosome 16 containing the wv gene and shown to occur in mutant but not in wild-type mice . We demonstrate tight linkage of the Girk2 mutation to the wv phenotype and refine the localization of the weaver (wv) gene on recombinational and physical maps . This linkage between Girk2 and wv has existed since at least 1988 in descendants of the original mutation maintained in C57BL/6 animals . Girk2 is shown to be transcribed in brain before the first recognized manifestation of the wv phenotype and in cultures of granule cells (GCs) isolated from cerebellum at postnatal day 8 . Wild-type GCs grown in this culture system display an important developmental property--the ability to extend neurites . However, no inwardly rectifying K+ current is detected in GCs cultured from either wv/wv or +/+ cerebellum under a variety of conditions that activate related channels in other tissues . This suggests that if the Girk2 mutation is responsible for the wv phenotype, it does not act by altering these electrical properties of developing GCs.

Chem Biol, 1995 Dec, 2(12), 847 - 55
Using evolutionary changes to achieve species-specific inhibition of enzyme action--studies with triosephosphate isomerase; Gomez-Puyou A et al.; BACKGROUND: Many studies that attempt to design species-specific drugs focus on differences in the three-dimensional structures of homologous enzymes . The structures of homologous enzymes are generally well conserved especially at the active site, but the amino-acid sequences are often very different . We reasoned that if a non-conserved amino acid is fundamental to the function or stability of an enzyme from one particular species, one should be able to inhibit only the enzyme from that species by using an inhibitor targeted to that residue . We set out to test this hypothesis in a model system . RESULTS: We first identified a non-conserved amino acid (Cys14) whose integrity is important for catalysis in triosephosphate isomerase (TIM) from Trypanosoma brucei . The equivalent residues in rabbit and yeast TIM are Met and Leu, respectively . A Cys14Leu mutant of trypanosomal TIM had a tendency to aggregate, reduced stability and altered kinetics . To model the effects of a molecule targeted to Cys14, we used methyl methanethiosulfonate (MMTS) to derivatize Cys14 to a methyl sulfide . This treatment dramatically inhibited TIMs with a Cys residue at a position equivalent to Cys14, but not rabbit TIM (20% inhibition) or yeast TIM (negligible inhibition), which lack this residue . CONCLUSIONS: Cys14 of trypanosomal TIM is a non-conserved amino acid whose alteration leads to loss of enzyme structure and function . TIMs that have a cysteine residue at position 14 could be selectively inhibited by MMTS . This approach may offer an alternative route to species-specific enzyme inhibition.

Curr Biol, 1995 Dec 1, 5(12), 1416 - 23
Physical interaction between a novel domain of the receptor Notch and the transcription factor RBP-J kappa/Su(H); Tamura K et al.; BACKGROUND: The mammalian transcription factor RBP-J kappa binds to the DNA sequence motif CGTGGGAA and is involved in the regulation of gene expression; for example, it plays a part in the transactivation of viral and cellular genes by Epstein-Barr virus nuclear antigen-2 . The Drosophila homologue of RBP-J kappa is the product of the Suppressor of Hairless (Su(H)) gene . Su(H) is a neurogenic gene that acts downstream of Notch, which encodes a cell-surface receptor . Furthermore, in the mouse, the phenotypes of homozygous mutant Notch1 embryos are very similar to those of homozygous mutant RBP-J kappa embryos . Recent studies, using the yeast two-hybrid system, have led to the suggestion that the CDC10/ankyrin-like repeats of the Drosophila Notch protein interact with the Su(H) protein . RESULTS: We searched for proteins that interact with mouse RBP-J kappa using the yeast two-hybrid system, and in this way identified a short intracellular region (mRAM23) of the mouse Notch1 protein that lacks any known sequence motif . In vitro interaction studies, using proteins fused to glutathione-S-transferase, showed that RBP-J kappa and Su(H) bind directly to the RAM23 regions of mouse Notch1 and Drosophila Notch, respectively . Immunoprecipitation analysis showed that RBP-J kappa and the mRAM23 region of mouse Notch1 also interact in vivo . Further studies, including site-directed mutagenesis experiments, narrowed down the region of mouse Notch1 that interacts with RBP-J kappa . The results indicate that this region is less than 50 amino-acid residues in length, and lies immediately downstream of the transmembrane region . CONCLUSIONS: We show that the transcription factor RBP-J kappa/Su(H) interacts directly with a novel intracellular domain of the cell-surface receptor Notch . RBP-J kappa/Su(H) does not appear to interact with Notch via the CDC10/ankyrin repeats implicated in previous studies.

Curr Biol, 1995 Dec 1, 5(12), 1404 - 15
A family of phosphoinositide 3-kinases in Drosophila identifies a new mediator of signal transduction; MacDougall LK et al.; BACKGROUND: Mammalian phosphoinositide 3-kinases (PI 3-kinases) are involved in receptor-mediated signal transduction and have been implicated in processes such as transformation and mitogenesis through their role in elevating cellular phosphatidylinositol (3,4,5)-trisphosphate . Additionally, a PI 3-kinase activity which generates phosphatidylinositol 3-phosphate has been shown to be required for protein trafficking in yeast . RESULTS: We have identified a family of three distinct PI 3-kinases in Drosophila, using an approach based on the polymerase chain reaction to amplify a region corresponding to the conserved catalytic domain of PI 3-kinases . One of these family members, PI3K_92D, is closely related to the prototypical PI 3-kinase, p110 alpha; PI3K_59F is homologous to Vps34p, whereas the third, PI3K_68D, is a novel PI 3-kinase which is widely expressed throughout the Drosophila life cycle . The PI3K_68D cDNA encodes a protein of 210 kDa, which lacks sequences implicated in linking p110 PI 3-kinases to p85 adaptor proteins, but contains an amino-terminal proline-rich sequence, which could bind to SH3 domains, and a carboxy-terminal C2 domain . Biochemical analyses demonstrate that PI3K_68D has a novel substrate specificity in vitro, restricted to phosphatidylinositol and phosphatidylinositol 4-phosphate, and is unable to phosphorylate phosphatidylinositol (4,5)-bisphosphate, the implied in vivo substrate for p110 . CONCLUSIONS: A family of PI 3-kinases in Drosophila, including a novel class represented by PI3K_68D, is described . PI3K_68D has the potential to bind to signalling molecules containing SH3 domains, lacks p85-adaptor-binding sequences, has a Ca(2+)-independent phospholipid-binding domain and displays a restricted in vitro substrate specificity, so it could define a novel signal transduction pathway.

Plant Cell, 1995 Dec, 7(12), 2151 - 61
Meiotic recombination break points resolve at high rates at the 5' end of a maize coding sequence; Xu X et al.; Sequence analysis of recombination break points has defined a 377-bp recombination hot spot within the anthocyanin 1 (a1) gene . One-fifth of all recombination events that occurred within the 140-kb a1-shrunken 2 interval resolved within this 377-bp hot spot . In yeast, meiotic double-strand breaks in chromosomal DNA are thought to initiate recombination and are generally located 5' of coding regions, near transcription promoter sequences . Because the a1 recombination hot spot is located within the 5' transcribed region of the a1 gene, the sites at which recombination events initiate and resolve appear to be different, but both appear to be regulated in relation to transcribed sequences . Although transposon insertions are known to suppress recombination and alter the ratio of crossovers to apparent gene conversions, the Mutator 1 transposon insertion in the a1-mum2 allele does not alter the sites at which recombination events resolve.

Zhongguo Zhong Yao Za Zhi, 1995 Dec, 20(12), 751 - 2, 764
{Antifebrile and anti-inflammatory effects of radix Cynanchi atrati}; Xue B et al.; The water extract of Radix Cynanchi Atrati used as intraperitoneal injection has been proved to have an obvious antifebrile++ effect on rat fever caused by 15% yeast suspension hypodermic injection as well as a significant anti-inflammatory effect . But the antiferbrile effect of its ethanol extract is not clear.

Hum Mol Genet, 1995 Dec, 4(12), 2363 - 71
Molecular genetic analysis of familial early-onset Alzheimer's disease linked to chromosome 14q24.3; Cruts M et al.; Genetic linkage studies have indicated that chromosome 14q24.3 harbours a major locus for early-onset (onset age <65 years) Alzheimer's disease (AD3) . Positional cloning efforts have identified a novel gene S182 or presenilin 1 as the AD3 gene . We have mapped S182 in the AD3 candidate region between D14S277 and D14S284 defined by genetic linkage studies in the two chromosome 14 linked, early-onset AD families AD/A and AD/B . We have shown that S182 is expressed in lymphoblasts and have determined the complete cDNA in both brain and lymphoblasts by RT-PCR sequencing . S182 is alternatively spliced in both brain and lymphoblasts within a putative phosphorylation site located 5' in the coding region . We identified two novel mutations, Ile143Thr and Gly384la located in, respectively, the second transmembrane domain and in the sixth hydrophilic loop of the putative transmembrane structure of S182 . As families AD/A and AD/B have very similar AD phenotype our observation of two mutations in functionally different domains suggest that onset age and severity of AD may not be very helpful predictors of the location of putative S182 mutations.

Farmaco, 1995 Dec, 50(12), 885 - 8
Chromonyl-aminosalicylic acid derivatives as possible antimycobacterial agents; Lacova M et al.; N-(2H,3H,4H-4-oxo-6-R1-2-R2O-benzopyran-3-ylidenmethyl) derivatives and imino derivatives of 3-, 4-, 5-aminosalicylic acids were prepared . It was found that some of the synthesized derivatives of 4-aminosalicylic acid are as effective against typical and atypical strains of mycobacteria as isoniazid (INH) . Interesting activity of some derivatives against yeast was also found.

Curr Opin Cell Biol, 1995 Dec, 7(6), 790 - 7
Starting the cell cycle: what's the point?
Cross FR.
Early work on regulation of the budding yeast cell cycle defined a critical regulatory step called 'Start', considered to represent cell cycle commitment . Recent work has defined the probable molecular basis of Start to be activation of Cln-Cdc28 protein kinase complexes . Cln-Cdc28 kinases may directly regulate many cell cycle processes, including some classically considered to be 'post-Start' . Specialization of function among the three genetically redundant CLN genes is becoming apparent.

Toxicol Lett, 1995 Dec, 82-83, 823 - 7
Genetically engineered mammalian cells and applications; Doehmer J et al.; In general, cells genetically engineered for stable and defined expression of xenobiotic-metabolizing enzymes are useful tools whenever a metabolism-related problem in toxicology and pharmacology is to be solved . It is the genetic and phenotypic nature of a given cell that determines its applicability . Mammalian cells have useful characteristics not given in bacterial, yeast or insect cells, which also may express xenobiotic-metabolizing enzymes . It is the problem to be solved and the question to be answered which determine the optimal choice for the best-suited expression system . There may even be subtle differences between mammalian cells of different species and organ origin, which might play a role in choosing a mammalian expression system . Thus, the level and specificity of the xenobiotic-metabolizing enzyme, the experimental testing conditions, and the biological endpoints present in a chosen cell are the most important criteria to be observed in the application of the mammalian expression systems.

Chromosome Res, 1995 Dec, 3(8), 497 - 506
Molecular characterization of a centromeric satellite DNA in the hemiclonal hybrid frog Rana esculenta and its parental species; Ragghianti M et al.; Hybrid water frogs Rana esculenta reproduce by hybridogenesis: one parental genome (of Rana lessonae) is excluded in the germ line, the other (of Rana ridibunda) is clonally transmitted to haploid gametes . The two parental species differ in that the amount of centromeric heterochromatin revealed by differential staining is much higher in Rana ridibunda . An abundant, tandemly arrayed, centromeric satellite DNA, designated RrS1, is revealed in Rana ridibunda genomes by the restriction endonuclease Stul, which generates a major repetitive sequence fragment of 300 and a minor one of 200 bp . This AT-rich (68%) satellite family is located at the centromeres of the five largest chromosomes (1-5) and of a medium to small heterobrachial one (8 or 9); it thus constitutes only part of the centromeric heterochromatin that characterizes all Rana ridibunda chromosomes . RrS1 represents about 2.5% of the genome of Rana ridibunda; it may represent as little as 0.2% of the genome of Rana lessonae, and cannot be detected in Xenopus laevis frogs or Salamandra salamandra and Triturus carnifex salamanders . Segments of the satellite sequence are similar to sequences of yeast centromeric DNA element CDEIII and of the mammalian CENP-B box . A role for RrS1 and other centromeric satellite DNAs in the germ line genome exclusion of the hybridogenetic frog hybrids, although suggested, has not yet been demonstrated.

Chromosome Res, 1995 Dec, 3(8), 473 - 8
Mapping of murine YACs containing the genes Cea2 and Cea4 after B1-PCR amplification and FISH-analysis; Rettenberger G et al.; PCR with primers specific for the murine B1 consensus sequence allows amplification of DNA from murine sources . We have used B1-PCR for amplifying yeast artificial chromosome (YAC) DNA which can be used to localize single YACs by fluorescence in situ hybridization . The genes for the pregnancy-specific glycoproteins Cea2 and Cea4, both belonging to the large carcinoembryonic antigen gene family, were localized by chromosomal in situ suppression hybridization of three YAC clones to murine chromosome 7A2-A3 . This was facilitated by the use of the mouse lymphoma cell line WMP/WMP which contains nine pairs of Robertsonian fusion chromosomes.

Am J Hum Genet, 1995 Dec, 57(6), 1351 - 63
A YAC contig encompassing the recessive Stargardt disease gene (STGD) on chromosome 1p; Anderson KL et al.; Stargardt disease (STGD) and fundus flavimaculatus are infrequent autosomal recessive conditions characterized by a juvenile macular dystrophy and variable degrees of peripheral retinal changes . Linkage analysis performed in 47 STGD/fundus flavimaculatus families demonstrated significant linkage to 13 polymorphic DNA markers on chromosome 1p . The maximum combined two-point lod score was 32.7 (maximum recombination fraction {phi max} = .006) with the polymorphic marker D1S188 . Our data demonstrate that STGD and fundus flavimaculatus are the same disorder clinically and genetically and provide further evidence for genetic homogeneity of this phenotype . Analysis of recombination events on disease chromosomes placed the STGD gene within a 4-cM interval between markers D1S435 and D1S236 . A physical map was constructed of a YAC contig flanking STGD, from markers D1S500 to D1S495, and includes the critical interval delineated by historical recombinants . This contig spans approximately 31 cM, with one gap (3-5 cM) that is outside the 4-cM critical region . Localization of STGD to a single YAC contig will facilitate its positional cloning.

Virology, 1995 Dec 1, 214(1), 59 - 71
Biochemical and genetic analyses of the interaction between the helicase-like and polymerase-like proteins of the brome mosaic virus; O'Reilly EK et al.; Replication of the three positive-strand genomic RNAs of brome mosaic virus requires the activities of the helicase-like 1a and the polymerase-like 2a proteins . One hundred fifteen amino acids of the 2a N-terminus and the 1a helicase-like region of over 50 kDa are both necessary and sufficient for 1a-2a interaction . Requirement of the large size of the 1a helicase-like domain suggests that higher order structures might be necessary for the protein's interaction with 2a . To explore the structural properties of 1a, we used limited proteolysis of in vitro-translated 1a protein . Treatment of 1a and its deletion derivatives with papain or trypsin revealed that the C-terminal helicase-like segment of approximately 50-60 kDa is highly resistant under our assay conditions to proteolysis, while the N-terminus is rapidly degraded . All tested mutations in the helicase-like region that renders this region protease-sensitive have previously been found to be defective for RNA replication in vivo . To complement the in vitro studies, we examined the interaction of the 1a helicase-like domain and the 2a N-terminus in yeast using the two-hybrid system . Mutations previously known to disrupt 1a-2a interaction also prevented interaction in yeast . Furthermore, results from two-hybrid analysis suggest that the structural domain mapped in vitro is important for 1a-2a interaction . Finally, we found that the helicase-like proteins of three other tripartite RNA viruses also contain equivalently located protease-resistant domains.

Virology, 1995 Dec 1, 214(1), 289 - 93
Dimerization of the human papillomavirus E7 oncoprotein in vivo; Clemens KE et al.; We have used a yeast two-hybrid system to show that human papillomavirus E7 proteins can form oligomeric complexes in vivo . The carboxyl-terminal cysteine-rich metal-binding domain is critical for this activity although amino-terminal sequences also contribute to oligomerization . Our experiments also reveal that E7 possesses an intrinsic transcription activation activity in yeast, which resides in the amino terminus of the protein.

J Cell Biol, 1995 Dec, 131(6 Pt 1), 1435 - 52
Rab 7: an important regulator of late endocytic membrane traffic; Feng Y et al.; Rab5 and rab7 proteins belong to a superfamily of small molecular weight GTPases known to be associated with early and late endosomes, respectively . The rab5 protein plays an important regulatory role in early endocytosis, yet the function of rab7 protein was previously uncharacterized . This question was addressed by comparing the kinetics of vesicular stomatitis virus (VSV) G protein internalization in baby hamster kidney cells overexpressing wild-type or dominant negative mutant forms of the rab7 protein (rab7N125I and rab7T22N) . Overexpression of wild-type rab7 protein allowed normal transport to late endosomes (mannose 6-phosphate receptor positive), while the rab7N125I mutant caused the VSV G protein to accumulate specifically in early (transferrin receptor positive) endosomes . Horseradish peroxidase and paramyxovirus SV5 hemagglutinin-neuraminidase (HN) were used in quantitative biochemical assays to further demonstrate that rab7 function was not required for early internalization events, but was crucial in downstream degradative events . The characteristic cleavage of SV5 HN in the late endosome distinguishes internalization from transport to later stages of the endocytic pathway . Mutant rab7N125I or rab7T22N proteins had no effect on the internalization of either horseradish peroxidase or SV5 HN protein . In contrast, the mutant proteins markedly inhibited the subsequent cleavage of the SV5 HN protein . Taken together, these data support a key role for rab7, downstream of rab5, in regulating membrane transport leading from early to late endosomes . We compare our findings to those obtained for the yeast homologues Ypt51p, Ypt52p, Ypt53p, and Ypt7p.

J Bacteriol, 1995 Dec, 177(24), 7050 - 9
Picrophilus gen . nov., fam . nov.: a novel aerobic, heterotrophic, thermoacidophilic genus and family comprising archaea capable of growth around pH 0; Schleper C et al.; Two species belonging to a novel genus of archaea, designated Picrophilus oshimae and Picrophilus torridus, have been isolated from two different solfataric locations in northern Japan . One habitat harboring both organisms was a dry, extremely acidic soil (pH < 0.5) that was heated by solfataric gases to about 55 degrees C . In the laboratory both species grew heterotrophically on yeast extract and poorly on tryptone under aerobic conditions at temperatures between 45 and 65 degrees C; they grew optimally at 60 degrees C . The pH optimum was 0.7, but growth occurred even around pH 0 . Under optimal conditions, the generation time was about 6 h, yielding densities of up to 10(10) cells per ml . The cells were surrounded by a highly filigreed regular tetragonal S-layer, and the core lipids of the membrane were mainly bis-phytanyltetraethers . The 16S rRNA sequences of the two species were about 3% different . The complete 16S rRNA sequence of P . oshimae was 9.3% different from that of the closest relative, Thermoplasma acidophilum . The morphology and physiological properties of the two species characterize Picrophilus as a novel genus that is a member of a novel family within the order Thermoplasmales.

Hum Genet, 1995 Dec, 96(6), 671 - 3
Human glial cell line-derived neurotrophic factor (GDNF) maps to chromosome 5; Bermingham N et al.; Neurotrophic factors are essential neurone survival promoting molecules that are often secreted and that bind to neuronal cell surface receptors . Glial cell line-derived neurotrophic factor, GDNF, is a potent neurotrophic factor that promotes the survival of dopaminergic neurones in cultures including embryonic neuronal cultures . We have mapped the gene encoding GDNF by two independent methods: using a cell hybrid panel and by fluorescent in situ hybridisation . We find GDNF lies on the short arm of human chromosome 5, at 5p13.1-p13.3 ability to promote dopamine uptake in midbrain cultures . The protein was partially sequenced and a rat GDNF cDNA was isolated by screening a B49 cDNA library with an oligonucleotide probe designed from the amino-terminus of the rat protein . Human GDNF sequences were isolated by screening a human genomic library with a portion of the rat GDNF cDNA (Lin et al . 1993) . We wished to localise the GDNF gene in the human genome and determine its proximity to possible sites of mutation, particularly phenotypes affecting neuronal function.

Hum Genet, 1995 Dec, 96(6), 655 - 60
Sperm nuclei analysis of a Robertsonian t(14q21q) carrier, by FISH, using three plasmids and two YAC probes; Rousseaux S et al.; The meiotic segregation of chromosomes 14 and 21 was analysed in 1116 spermatozoa from an oligoasthenospermic carrier of a Robertsonian translocation t(14q21q), and in 16,392 spermatozoa from a control donor, using two-colour fluorescence in situ hybridisation (FISH) . Two YAC probes (cloned in yeast artificial chromosomes) specific for regions on the long arms of these chromosomes were co-hybridised . Of the spermatozoa, 12% were unbalanced, resulting from adjacent segregations . Chromosomes X, Y and 1 were also simultaneously detected in 1335 spermatozoa from the same carrier . Whereas gonosomal disomy rates were not significantly different from those of the control donors, disomy 1 were slightly but significantly increased to 0.7% . The diploidy rate was also slightly increased to approximately 1% in the translocation carrier.

J Bacteriol, 1995 Dec, 177(23), 6937 - 45
A novel endo-beta-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum; de la Cruz J et al.; The mycoparasitic fungus Trichoderma harzianum CECT 2413 produces at least three extracellular beta-1,3-glucanases . The most basic of these extracellular enzymes, named BGN13.1, was expressed when either fungal cell wall polymers or autoclaved mycelia from different fungi were used as the carbon source . BGN13.1 was purified to electrophoretic homogeneity and was biochemically characterized . The enzyme was specific for beta-1,3 linkages and has an endolytic mode of action . A synthetic oligonucleotide primer based on the sequence of an internal peptide was designed to clone the cDNA corresponding to BGN13.1 . The deduced amino acid sequence predicted a molecular mass of 78 kDa for the mature protein . Analysis of the amino acid sequence indicates that the enzyme contains three regions, one N-terminal leader sequence; another, nondefined sequence; and one cysteine-rich C-terminal sequence . Sequence comparison shows that this beta-1,3-glucanase, first described for filamentous fungi, belongs to a family different from that of its previously described bacterial, yeast, and plant counterparts . Enzymatic-activity, protein, and mRNA data indicated that bgn13.1 is repressed by glucose and induced by either fungal cell wall polymers or autoclaved yeast cells and mycelia . Finally, experimental evidence showed that the enzyme hydrolyzes yeast and fungal cell walls.

Cancer Res, 1995 Dec 1, 55(23), 5574 - 9
Inhibition of CYP1A2 and CYP3A4 by oltipraz results in reduction of aflatoxin B1 metabolism in human hepatocytes in primary culture; Langouet S et al.; Dithiolethiones are thought to act as potent chemoprotective agents against aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat by inducing glutathione S-transferases (GSTs) . To determine whether these antioxidants can be similarly effective in human beings, we have investigated metabolism of AFB1, in primary human hepatocytes with or without pretreatment by oltipraz (OPZ), a synthetic derivative of the natural 1,2-dithiole-3-thione . Aflatoxin M1 (AFM1), glutathione conjugates of AFB1 oxides (AFBSGs), and unchanged AFB1 were quantitated in cultures derived from eight human liver donors . Parenchymal cells obtained from the three GST M1-positive livers metabolized AFB1 to AFM1 and to AFBSGs derived from the isomeric exo-and endo-8,9-oxides, whereas no AFBSGs were formed in the GST M1-null cells . Pretreatment of the cells with 3-methylcholanthrene or rifampicin, inducers of CYP1A2 and CYP3A4, respectively, caused a significant increase in AFB1 metabolism . Although OPZ induced GST A2, and to a lesser extent GST A1 and GST M1, it decreased formation of AFM1 and AFBSG, which involves CYP1A2 and CYP3A4 . Inhibition by OPZ of AFB1 metabolism by reducing CYP1A2 and CYP3A4 was also demonstrated by decreased activity of their monooxygenase activities toward ethoxyresorufin and nifedipine, respectively . The significant inhibition by OPZ of human recombinant yeast CYP1A2 and CYP3A4 was also shown . These results demonstrate that AFBSG can be formed by GST M1-positive human hepatocytes only, and suggest that chemoprotection with OPZ is due to an inhibition of activation of AFB1, in addition to a GST-dependent inactivation of the carcinogenic exo-epoxide.

Cancer Res, 1995 Dec 1, 55(23), 5504 - 6
Human homologue of a candidate for the Mom1 locus, the secretory type II phospholipase A2 (PLA2S-II), maps to 1p35-36.1/D1S199; Praml C et al.; Mice heterozygous for the dominant Min mutation in their Apc gene develop multiple intestinal neoplasia . Analogously, family members from familial adenomatous polyposis kindreds inheriting mutations in their human APC homologue develop a similar phenotype . Quantitative trait loci studies have identified the Mom1 locus (for modifier of Min-1), which is responsible for part of the genetic variability in polyp number found among inbred mouse strains . The secretory type II phospholipase {nonpancreatic Pla2s (type II Pla2s or Pla2s-II)} has been demonstrated to be a candidate for Mom1, and a mutation in Pla2s-II in mice carrying the Min mutation has been proposed to account for an increased polyp number compared to mice without the Pla2s-II mutation . In this study, we have mapped the chromosomal position of the human homologue of Pla2s-II . We have identified 3 mega-yeast artificial chromosomes that carry PLA2S-II and localized one of them by fluorescence in situ hybridization to the border between 1p35 and 1p36.1 . The presence of the microsatellite marker D1S199 in all three clones integrates PLA2S-II into different genetic maps . This highly polymorphic CA repeat D1S199 has previously been shown by us to identify loss of heterozygosity in 48% of sporadic colorectal tumors, indicating that the human homologue of the Pla2s-II/Mom1 locus might be related to human colorectal cancer.

Arch Biochem Biophys, 1995 Dec 1, 324(1), 9 - 14
Application of a double isotopic labeling method to a study of the interaction of mitochondrially bound rat brain hexokinase with intramitochondrial compartments of ATP generated by oxidative phosphorylation; de Cerqueira Cesar M et al.; gamma-Labeled ATP was produced by rat brain mitochondria utilizing {32P}Pi as substrate for oxidative phosphorylation . The 32P/14C ratio of Glc-6-P produced by the endogenous mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) using {U-14C}Glc as substrate was determined as a function of time after initiation of oxidative phosphorylation . This same ratio was determined for Glc-6-P formed by added yeast hexokinase using extramitochondrial ATP as substrate . The specific activity of ATP formed by oxidative phosphorylation was manipulated either by initiating the reaction with labeled Pi and subsequently adding excess unlabeled Pi or by initiating the reaction with unlabeled Pi and introducing the labeled substrate at a later time . The 32P/14C ratio of Glc-6-P formed by yeast hexokinase, reflecting the specific activity of ATP in the extramitochondrial space, was rapidly responsive to such manipulations, but the corresponding changes in the 32P/14C ratio of Glc-6-P produced by the endogenous hexokinase were markedly different . The results are consistent with the view that mitochondrially bound hexokinase does not utilize extramitochondrial ATP as substrate but rather is functionally coupled to a discrete intramitochondrial compartment of ATP produced by oxidative phosphorylation.

J Biol Chem, 1995 Dec 1, 270(48), 28982 - 8
Cloning of a novel family of mammalian GTP-binding proteins (RagA, RagBs, RagB1) with remote similarity to the Ras-related GTPases; Schurmann A et al.; cDNA clones of two novel Ras-related GTP-binding proteins (RagA and RagB) were isolated from rat and human cDNA libraries . Their deduced amino acid sequences comprise four of the six known conserved GTP-binding motifs (PM1, -2, -3, G1), the remaining two (G2, G3) being strikingly different from those of the Ras family, and an unusually large C-terminal domain (100 amino acids) presumably unrelated to GTP binding . RagA and RagB differ by seven conservative amino acid substitutions (98% identity), and by 33 additional residues at the N terminus of RagB . In addition, two isoforms of RagB (RagBs and RagB1) were found that differed only by an insertion of 28 codons between the GTP-binding motifs PM2 and PM3, apparently generated by alternative mRNA splicing . Polymerase chain reaction amplification with specific primers indicated that both long and short form of RagB transcripts were present in adrenal gland, thymus, spleen, and kidney, whereas in brain, only the long form RagB1 was detected . A long splicing variant of RagA was not detected . Recombinant glutathione S-transferase (GST) fusion proteins of RagA and RagBs bound large amounts of radiolabeled GTP gamma S in a specific and saturable manner . In contrast, GTP gamma S binding of GST-RagB1 hardly exceeded that of recombinant GST . GTP gamma S bound to recombinant RagA, and RagBs was rapidly exchangeable for GTP, whereas no intrinsic GTPase activity was detected . A multiple sequence alignment indicated that RagA and RagB cannot be assigned to any of the known subfamilies of Ras-related GTPases but exhibit a 52% identity with a yeast protein (Gtr1) presumably involved in phosphate transport and/or cell growth . It is suggested that RagA and RagB are the mammalian homologues of Gtr1 and that they represent a novel subfamily of Ras-homologous GTP binding proteins.

Nat Genet, 1995 Dec, 11(4), 395 - 401
Peroxisome assembly factor-2, a putative ATPase cloned by functional complementation on a peroxisome-deficient mammalian cell mutant; Tsukamoto T et al.; Rat peroxisome assembly factor-2 (PAF-2) cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP92, using transient transfection assay . This cDNA encodes a 978-amino acid protein with two putative ATP-binding sites . PAF-2 is a member of a putative ATPase family, including two yeast gene products essential for peroxisome assembly . A stable transformant of ZP92 with the cDNA was morphologically and biochemically restored for peroxisome biogenesis . Fibroblasts derived from patients deficient in peroxisome biogenesis (complementation group C) were also complemented with PAF-2 cDNA, indicating that PAF-2 is a strong candidate for the pathogenic gene of group C peroxisome deficiency.

Surgery, 1995 Dec, 118(6), 1018 - 23
Molecular and cytogenetic characterization of a t(1;10;21) translocation in the human papillary thyroid cancer cell line TPC-1 expressing the ret/H4 chimeric transcript; Jossart GH et al.; BACKGROUND . Activation of the ret proto-oncogene by three different chromosomal rearrangements occurs in up to 25% of papillary thyroid carcinomas . We developed a rapid screening technique to detect ret rearrangements in human interphase and metaphase cells on the basis of multicolor fluorescence in situ hybridization (FISH) of locus-specific DNA probes . METHODS . DNA from individual clones representing the respective ends of a yeast artificial chromosome (YAC) contig spanning the entire ret gene locus were labeled with either digoxigenin (visualized in red) or biotin (green) and hybridized to normal human lymphocytes and the papillary thyroid cancer cell line TPC-1 expressing the ret/H4 chimeric transcript . Further detailed analysis was performed with whole chromosome painting probes and locus-specific probes (YACs, P1s, DNA repeat probes) on tumor metaphase spreads . RESULTS . Hybridization of the YACs to unrearranged ret loci in normal human lymphocyte interphase nuclei showed two yellow domains because of probe overlap . Hybridization to TPC-1 interphase nuclei showed one yellow domain, and 1 red and 1 green domain separated by a large physical distance . Further analysis of metaphase spreads revealed a complex translocation t(1;10;21)(1pter > 1q31::21q22.1 > 21qter; 10q11.2 > 10pter::1q31 > 1qter; 21pter > 21q22.1;;10q21.2 > 10q11.2::10q21.2 > 10qter) and loss of the H4 gene locus on the nontranslocated chromosome 10 . CONCLUSIONS . Break point spanning probes can reliably detect ret rearrangements in interphase nuclei . Locus-specific and whole chromosome painting probes can be used to further characterize complex rearrangements by fluorescence in situ hybridization to metaphase spreads . The papillary thyroid cancer cell line TPC-1 carries the paracentric inversion 10q, inv(10)(q11.2q21) and a complex t(1; 10; 21) translocation . Deletion of the H4 gene on the chromosome 10 not involved in the t(1; 10; 21) translocation suggests lack of normal H4 expression in the TPC-1 cell line . Further studies will have to address the role of the H4 gene product in tumor genesis and progression.

Science, 1995 Dec 1, 270(5241), 1464 - 72
Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog; Nissen P et al.; The structure of the ternary complex consisting of yeast phenylalanyl-transfer RNA (Phe-tRNAPhe), Thermus aquaticus elongation factor Tu (EF-Tu), and the guanosine triphosphate (GTP) analog GDPNP was determined by x-ray crystallography at 2.7 angstrom resolution . The ternary complex participates in placing the amino acids in their correct order when messenger RNA is translated into a protein sequence on the ribosome . The EF-Tu-GDPNP component binds to one side of the acceptor helix of Phe-tRNAPhe involving all three domains of EF-Tu . Binding sites for the phenylalanylated CCA end and the phosphorylated 5' end are located at domain interfaces, whereas the T stem interacts with the surface of the beta-barrel domain 3 . The binding involves many conserved residues in EF-Tu . The overall shape of the ternary complex is similar to that of the translocation factor, EF-G-GDP, and this suggests a novel mechanism involving "molecular mimicry" in the translational apparatus.

J Biol Chem, 1995 Nov 24, 270(47), 28018 - 21
Increased drug affinity as the mechanistic basis for drug hypersensitivity of a mutant type II topoisomerase; Froelich-Ammon SJ et al.; Altered sensitivity of topoisomerase II to anticancer drugs profoundly affects the response of eukaryotic cells to these agents . Therefore, several approaches were employed to elucidate the mechanism of drug hypersensitivity of the mutant yeast type II topoisomerase, top2H1012Y . This mutant, which is approximately 5-fold hypersensitive to ellipticine, formed DNA cleavage complexes more rapidly than the wild-type yeast enzyme in the presence of the drug . Conversely, no change in the rate of DNA religation was observed . There was, however, a correlation between increased cleavage rates and enhanced drug binding affinity . The apparent dissociation constant for ellipticine in the mutant topoisomerase II.drug.DNA ternary complex was approximately 5-fold lower than in the wild-type ternary complex . Furthermore, the apparent KD value for the mutant binary (topoisomerase II.drug) complex was approximately 2-fold lower than the corresponding wild-type complex, indicating that drug hypersensitivity is intrinsic to the enzyme . These findings strongly suggest that the enhanced ellipticine binding affinity for topoisomerase II is the mechanistic basis for drug hypersensitivity of top2H1012Y.

Science, 1995 Nov 24, 270(5240), 1354 - 7
Sequence and characterization of a coactivator for the steroid hormone receptor superfamily; Onate SA et al.; A yeast two-hybrid system was used to identify a protein that interacts with and enhances the human progesterone receptor (hPR) transcriptional activity without altering the basal activity of the promoter . Because the protein stimulated transactivation of all the steroid receptors tested, it has been termed steroid receptor coactivator-1 (SRC-1) . Coexpression of SRC-1 reversed the ability of the estrogen receptor to squelch activation by hPR . Also, the amino terminal truncated form of SRC-1 acted as a dominant-negative repressor . Together, these results indicate that SRC-1 encodes a coactivator that is required for full transcriptional activity of the steroid receptor superfamily.

Proc Natl Acad Sci U S A, 1995 Nov 21, 92(24), 10849 - 53
Mapping the mouse genome: current status and future prospects; Dietrich WF et al.; The mouse is the best model system for the study of mammalian genetics and physiology . Because of the feasibility and importance of studying genetic crosses, the mouse genetic map has received tremendous attention in recent years . It currently contains over 14,000 genetically mapped markers, including 700 mutant loci, 3500 genes, and 6500 simple sequence length polymorphisms (SSLPs) . The mutant loci and genes allow insights and correlations concerning physiology and development . The SSLPs provide highly polymorphic anchor points that allow inheritance to be traced in any cross and provide a scaffold for assembling physical maps . Adequate physical mapping resources--notably large-insert yeast artificial chromosome (YAC) libraries--are available to support positional cloning projects based on the genetic map, but a comprehensive physical map is still a few years away . Large-scale sequencing efforts have not yet begun in mouse, but comparative sequence analysis between mouse and human is likely to provide tremendous information about gene structure and regulation.

Proc Natl Acad Sci U S A, 1995 Nov 21, 92(24), 10836 - 40
The genome of Caenorhabditis elegans; Waterston R et al.; The physical map of the 100-Mb Caenorhabditis elegans genome consists of 17,500 cosmids and 3500 yeast artificial chromosomes (YACs) . A total of 22.5 Mb has been sequenced, with the remainder expected by 1998 . A further 15.5 Mb of unfinished sequence is freely available online: because the areas sequenced so far are relatively gene rich, about half the 13,000 genes can now be scanned . More than a quarter of the genes are represented by expressed sequence tags (ESTs) . All information pertaining to the genome is publicly available in the ACeDB data base.

Proc Natl Acad Sci U S A, 1995 Nov 21, 92(24), 10831 - 5
The genome of Arabidopsis thaliana; Goodman HM et al.; Arabidopsis thaliana is a small flowering plant that is a member of the family cruciferae . It has many characteristics--diploid genetics, rapid growth cycle, relatively low repetitive DNA content, and small genome size--that recommend it as the model for a plant genome project . The current status of the genetic and physical maps, as well as efforts to sequence the genome, are presented . Examples are given of genes isolated by using map-based cloning . The importance of the Arabidopsis project for plant biology in general is discussed.

Genomics, 1995 Nov 20, 30(2), 376 - 9
Localization of cDNAs to a region poorly represented in the CEPH chromosome 21 YAC contig: candidate genes for genetic diseases mapped to 21q22.3; Gardiner K et al.; Fifty-three cDNA fragments previously obtained by hybridization selection from random clones in the chromosome 21 cosmid library LL21CNO2 failed to identify clones in the chromosome 21 YAC contig described by Chumakov et al . (1992, Nature 359: 380-387) . Using an expanded panel of somatic cell hybrids, we have verified that the majority of these cDNAs map to chromosome 21 and that in particular a very high proportion, approximately 85%, localize to a 5-Mb region of distal 21q22.3 . Pulsed-field analysis coupled with information from the NotI restriction map of the region further indicate that 17 cDNA fragments map within 650 kb of the PFKL gene and thus may be candidates for genetic diseases linked to this gene . This work helps to characterize a region poorly represented in the CEPH YAC contig and adds to the number of cDNAs useful in analysis of chromosome 21-associated diseases.

Genomics, 1995 Nov 20, 30(2), 372 - 5
The interferon-inducible, double-stranded RNA-specific adenosine deaminase gene (DSRAD) maps to human chromosome 1q21.1-21.2; Weier HU et al.; The interferon-inducible double-stranded RNA-specific adenosine deaminase is an RNA-modifying enzyme implicated in the generation of biased hypermutations viral RNAs and the site-selective editing of mammalian mRNAs of neural origin . The gene for the dsRNA-specific adenosine deaminase has been mapped by fluorescence in situ hybridization (FISH) of genomic clones to a single locus on human chromosome 1 bands q21.1-21.2 . Simultaneous multicolor FISH including lambda clones and yeast artificial chromosomes showed a localization of the gene in band 1q21 centromeric of D1S1705.

Genomics, 1995 Nov 20, 30(2), 347 - 9
Mapping of the human dentin matrix acidic phosphoprotein gene (DMP1) to the dentinogenesis imperfecta type II critical region at chromosome 4q21; Aplin HM et al.; Dentinogenesis imperfecta type II (DGI1) is an autosomal dominant disorder of dentin formation, which has been mapped to human chromosome 4q12-q21 . The region most likely to contain the DGI1 locus is a 3.2-cM region surrounding the osteopontin (SPP1) locus . Recently, a novel dentin-specific acidic phosphoprotein (dmp1) has been cloned in the rat and mapped to mouse chromosome 5q21 . In the current investigation, we have isolated a cosmid containing the human DMP1 gene . The isolation of a short tandem repeat polymorphism at this locus has allowed us to map the DMP1 locus to human chromosome 4q21 and demonstrate that it is tightly linked to DGI1 in two families (Zmax = 11.01, theta = 0.001) . The creation of a yeast artificial chromosome contig around SPP1 has further allowed us to demonstrate that DMP1 is located within 150 kb of the bone sialoprotein and 490 kb of the SPP1 loci, respectively . DMP1 is therefore a strong candidate for the DGI1 locus.

Genomics, 1995 Nov 20, 30(2), 251 - 6
LIS2, gene and pseudogene, homologous to LIS1 (lissencephaly 1), located on the short and long arms of chromosome 2; Reiner O et al.; We report here the isolation of a novel cDNA, designated LIS2, that maps to chromosome 2p11.2 by in situ hybridization and demonstrates extremely high sequence similarity to the recently identified LIS1 gene involved in Miller-Dieker lissencephaly at 17p13.3 . Specific probes for LIS2 revealed a pattern of expression resembling that of LIS1, although LIS2 is less abundant . Surprisingly, LIS2 detected an additional, higher molecular weight transcript in adult skeletal muscle . Isolated YAC clones and P1 clones mapped by in situ hybridization to two loci on chromosome 2,2p11.2 and 2q13-q14 . This hybridization was due to the existence of LIS2 pseudogene LIS2P on the long arm of chromosome 2.

Genomics, 1995 Nov 20, 30(2), 149 - 56
A novel Creb family gene telomeric of HLA-DRA in the HLA complex; Min J et al.; cDNA selection was used to identify genes encoded by a 440-kb yeast artificial chromosome (YAC) clone that spanned from HLA-DRA to CYP21 in the HLA complex . An initially selected short cDNA was used to isolate a 2639-nucleotide, apparently full-length cDNA from a human tonsil library . This cDNA contained one extended open reading frame that predicted a protein of 700 amino acids with a basic region and a leucine zipper that is highly similar to members of the Creb/ATF subfamily . High-stringency Southern blotting of total human genomic DNA using this cDNA as the probe showed only a single locus that mapped to the selecting YAC clone . This gene, designated Creb-related protein (Creb-rp), is expressed ubiquitously and is evolutionarily conserved in mammals . It is located in the HLA Class III region 6-10 kb centromeric of the XB gene, which encodes a tenascin-like extracellular matrix protein . Homologous sequences are located in the Class II-Class III interval of the mouse H-2 complex . The amino acid sequence homology and general structural features of the predicted protein indicate that this gene encodes a general transcription factor belonging to the Creb/ATF subfamily of the bZip super-family.

Gene, 1995 Nov 20, 165(2), 155 - 61
Construction of a cDNA library for a specific region of a chromosome using a novel cDNA selection method utilizing latex particles; Hayashida N et al.; A novel method is described for the rapid concentration of particular cDNAs and their mapping to specific regions of a genome . The strategy for 'cDNA scanning' is based on the hybridization of an entire library of cDNAs to a large fragment of genomic DNA that is covalently bound to latex particles . The hybridized cDNAs are eluted, amplified by PCR and cloned into a lambda vector . Selected cDNAs that hybridized to the genomic DNA are cloned, with subsequent sequence analysis . Region-specific DNA fragments prepared from a yeast artificial chromosome (YAC) clone, EG10D9, which maps to chromosome 5 of the small cruciferous plant Arabidopsis thaliana (At), were used to prepare a model system and were covalently bound to latex particles . cDNAs that hybridized to EG10D9 were concentrated by hybridization to the immobilized DNA . The hybridized cDNAs were recovered and amplified by PCR . The resultant sub-library of cDNAs of 0.5-2 kb in length was enriched about 1000-fold . The partial sequences of the cDNAs provided information about genes that are located on the EG10D9 region of the At genome . The cDNA scanning strategy provides an efficient method for the mapping of expressed genes which could be used as expressed sequence tags (EST) within a genome.

J Biol Chem, 1995 Nov 17, 270(46), 27419 - 22
Ionizable P1 residues in serine proteinase inhibitors undergo large pK shifts on complex formation; Abul Qasim M et al.; The burial of charged residues in proteins is rare as it is thermodynamically strongly disfavored . However, in "standard mechanism" protein inhibitors of serine proteinases, the P1 residue, which is highly exposed, becomes buried in the S1 specificity pocket of the enzyme . In many enzymes, such as Streptomyces griseus proteinase B (SGPB) the S1 pocket is hydrophobic . We measured the pH dependence of the association equilibrium constant for the interaction of SGPB with turkey ovomucoid third domain P1 mutants, Glu18 OMTKY3 and His18 OMTKY3 . In order to eliminate the effects of other ionizable groups on the enzyme and the inhibitor, we divided these pH dependences by the pH dependence of the association equilibrium constant for the Gln18 OMTKY3 mutant . This yielded for Glu18, pKf (free inhibitor) of 4.46 +/- 0.05 and pKc (complex) of 8.74 +/- 0.06 . For His18 the values are pKf 6.63 +/- 0.08 and pKc 4.31 +/- 0.07 . At low pH values Glu18 variant is a relatively good inhibitor for SGPB . This may be biologically relevant.

J Biol Chem, 1995 Nov 17, 270(46), 27395 - 8
Thrombin induces activation of p38 MAP kinase in human platelets; Kramer RM et al.; In human platelets a proline-directed kinase distinct from the ERK MAP kinases is stimulated by both thrombin and the thrombin receptor agonist peptide SFLLRN and may be involved in the activation of Ca(2+)-dependent cytosolic phospholipase A2 (Kramer, R . M., Roberts, E . F., Hyslop, P . A., Utterback, B . G., Hui, K . Y., and Jakubowski, J.A . (1995) J . Biol . Chem . 270, 14816-14823) . Here we show that this kinase is identical with or closely related to p38 (the mammalian homolog of HOG1 from yeast), a recently discovered protein kinase typically activated by inflammatory cytokines and environmental stress . Further, we demonstrate that activation of this kinase by thrombin is transient (with maximal stimulation at 1 min), is accompanied by tyrosine phosphorylation, and precedes the activation of the ERK kinases . This is the first report to show that p38 kinase is activated by thrombin and to suggest a role for this MAP kinase in the thrombin-mediated signaling events during platelet activation.

Eur J Biochem, 1995 Nov 15, 234(1), 200 - 7
A novel serine/threonine-specific protein phosphotransferase activity of Nm23/nucleoside-diphosphate kinase; Engel M et al.; Two human nm23 genes have been identified, designated nm23-H1 and nm23-H2, which encode the 88% identical nucleoside-diphosphate kinase (NDPK) A and NDPK B polypeptides, respectively . The nm23-H1 gene product has been shown to play a functional role in the suppression of tumor metastasis . The Nm23 proteins/NDPK are highly conserved throughout evolution and are implicated in controlling cellular differentiation and development in various species, while the underlying mechanisms remain undefined . Neither the NDPK activity nor the DNA-binding activity, identified recently for NDPK B, can satisfactory explain the regulatory functions of Nm23 . The present study provides evidence that purified Nm23 proteins are capable of transferring a phosphate group to other proteins when non-denaturing amounts of urea are present . This novel Nm23/NDPK activity was found to be specific for serine and threonine residues, and the transphosphorylation of substrate proteins occurred stoichiometrically . Because of the absence of a substrate turn-over, the novel function was termed protein phosphotransferase activity instead of protein kinase activity . It is demonstrated that urea stimulates the interaction of NDPK with other proteins . Identical phosphoprotein patterns were obtained using purified NDPK preparations from human, Drosophila, yeast and Dictyostelium in the presence of urea . Partially purified NDPK from human erythrocytes produced a similar phosphorylation pattern independent of urea addition and also acted stoichiometrically . In this preparation, a protein phosphotransferase activity of Nm23 species may possibly be generated and/or stabilized by the interaction with copurified proteins . Using different mutants of Dictyostelium NDPK it was shown that the protein phosphotransferase activity depends on the same active site as the NDPK activity . A phosphotransfer mechanism analogous to that of protein-histidine kinases is proposed, involving a high-energy phosphohistidine intermediate . Furthermore, the novel Nm23 function is compared with an apparently similar protein phosphotransferase activity which was observed previously with partially purified NDPK from different plant species.

Blood, 1995 Nov 15, 86(10), 3640 - 7
The myeloid zinc finger gene, MZF-1, regulates the CD34 promoter in vitro; Morris JF et al.; MZF-1 is a C2H2 zinc finger gene encoding a putative transcriptional regulator of myeloid differentiation . The MZF-1 protein contains 13 C2H2 zinc fingers arranged in bipartite DNA binding domains containing zinc fingers through 4 and, in the carboxy-terminus, 5 through 13 . We previously identified the DNA consensus binding site recognized by the two DNA binding domains . To assess the transcription regulatory function of MZF-1, the full-length MZF-1 coding region was fused to the DNA binding domain of the yeast transactivator GAL4 . The expression vector was cotransfected with the chloramphenicol acetyl transferase (CAT) reporter gene regulated by the thymidine kinase promoter containing GAL4 DNA binding sites into NIH 3T3, 293, K562, and Jurkat cell lines . MZF-1 represses CAT reporter gene expression via GAL4 binding sites in the nonhematopoietic cell lines NIH 3T3 and 293 . In contrast, MZF-1 activates CAT reporter gene expression in the hematopoietic cell lines K562 and Jurkat . The MZF-1 binding sites are present in the promoters of several genes expressed during myeloid differentiation, including the CD34 promoter . MZF-1 transcriptional regulation of this physiologically relevant promoter was assessed in both hematopoietic and nonhematopoietic cell lines . Recombinant MZF-1 protein specifically binds to the consensus binding sites in the CD34 promoter in mobility shift assays . MZF-1 expression vectors were cotransfected with the luciferase reporter plasmids regulated by the CD34 promoter into both nonhematopoietic and hematopoietic cell lines . As with the heterologous DNA binding domain, MZF-1 represses reporter gene expression in nonhematopoietic cell lines and activates expression in hematopoietic cell lines . Activation of CD34 expression in hematopoietic cell lines is dependent on the presence of intact MZF-1 binding sites . The cell type-specific regulation of the CD34 promoter by MZF-1 suggests the presence of tissue-specific regulators/adapters or differential MZF-1 modifications that determine MZF-1 transcriptional regulatory function.

Biochem J, 1995 Nov 15, 312 ( Pt 1), 17 - 21
Expression cloning of a zinc-finger cyclic AMP-response-element-binding protein; O'Brien RM et al.; In response to specific extracellular signals, intracellular cyclic AMP levels increase, leading to a variety of responses including the alteration of transcription of many eukaryotic genes . This transcriptional effect is frequently mediated through the cyclic AMP-response element (CRE) motif T(T/G)ACGTCA . Using an expression screening approach we have cloned a yeast gene, MSN2, that encodes a 78 kDa protein that recognizes this consensus CRE motif . Phosphorylation of the MSN2 protein by the catalytic subunit of protein kinase A stimulates DNA binding in vitro . Two putative Cys2His2-type zinc fingers present in the C-terminal 79 amino acids of the MSN2 protein are sufficient to confer CRE-binding specificity . Therefore, MSN2 represents a novel CRE-binding protein distinct from the multiple previously characterized basic region-leucine zipper repeat CRE-binding proteins.

Biochem Biophys Res Commun, 1995 Nov 13, 216(2), 630 - 5
Fine mapping of MEP1A, the gene encoding the alpha subunit of the metalloendopeptidase meprin, to human chromosome 6P21; Jiang W et al.; Meprins are kidney and intestinal proteases encoded by two distinct genes, MEP1A and MEP1B . MEP1A was previously mapped to human chromosome 6p, by the use of radiation and somatic cell hybrids, in the region containing the gene for autosomal recessive polycystic kidney disease (ARPKD) . We now report the fine mapping of MEP1A using yeast artificial chromosome clones, and linkage analysis of ARPKD families . The results from both physical and genetic mapping exclude MEP1A as a candidate for ARPKD . These studies place MEP1A in a region more telomeric to 6p12 and closer to the HLA loci than previously reported . More specifically, MEP1A is localized between loci D6S272 and D6S282, close to D6S452, on human chromosome 6p21.2-p21.1 . The more precise location of MEP1A will facilitate genetic studies of this locus and clarify the relation of this gene to others.

Gene, 1995 Nov 7, 165(1), 109 - 13
Cloning and sequencing of the beta-glucosidase-encoding gene from Candida molischiana strain 35M5N; Janbon G et al.; We have isolated a gene (bgln) encoding beta-glucosidase (beta Glu) from a cosmid library of the yeast, Candida molischiana 35M5N . The nucleotide sequence of bgln and its flanking regions was determined . This gene was found to be composed of 2289 bp and 763 amino acid (aa) residues encoding an 83.3-kDa protein . The aa sequence shared eleven putative N-glycosylation sites . Homology comparisons showed that this enzyme can be considered as a new member of the family-3 glycosyl hydrolases . Multiple alignment experiments revealed four conserved regions on aa sequences from beta Glu of this family.

Proc Natl Acad Sci U S A, 1995 Nov 7, 92(23), 10531 - 4
Mxi2, a mitogen-activated protein kinase that recognizes and phosphorylates Max protein; Zervos AS et al.; We describe Mxi2, a human protein that interacts with Max protein, the heterodimeric partner of the Myc oncoprotein . Mxi2 encodes a 297-residue protein whose sequence indicates that it is related to extracellular signal-regulated kinases (ERK protein kinases) . Mxi2 in yeast interacts with Max and with the C terminus of c-Myc . Mxi2 phosphorylates Max both in vitro and in vivo . The Mxi2 putative substrate recognition region has sequence similarity to the helix-loop-helix region in Max and c-Myc, suggesting that substrate recognition might be mediated via this motif . Phosphorylation by Mxi2 may affect the ability of Max to oligomerize with itself and its partners, bind DNA, or regulate gene expression.

Proc Natl Acad Sci U S A, 1995 Nov 7, 92(23), 10467 - 71
Molecular cloning and characterization of a cellular phosphoprotein that interacts with a conserved C-terminal domain of adenovirus E1A involved in negative modulation of oncogenic transformation; Schaeper U et al.; The adenovirus type 2/5 E1A proteins transform primary baby rat kidney (BRK) cells in cooperation with the activated Ras (T24 ras) oncoprotein . The N-terminal half of E1A (exon 1) is essential for this transformation activity . While the C-terminal half of E1A (exon 2) is dispensable, a region located between residues 225 and 238 of the 243R E1A protein negatively modulates in vitro T24 ras cooperative transformation as well as the tumorigenic potential of E1A/T24 ras-transformed cells . The same C-terminal domain is also required for binding of a cellular 48-kDa phosphoprotein, C-terminal binding protein (CtBP) . We have cloned the cDNA for CtBP via yeast two-hybrid interaction cloning . The cDNA encodes a 439-amino acid (48 kDa) protein that specifically interacts with exon 2 in yeast two-hybrid, in vitro protein binding, and in vivo coimmunoprecipitation analyses . This protein requires residues 225-238 of the 243R E1A protein for interaction . The predicted protein sequence of the isolated cDNA is identical to amino acid sequences obtained from peptides prepared from biochemically purified CtBP . Fine mapping of the CtBP-binding domain revealed that a 6-amino acid motif highly conserved among the E1A proteins of various human and animal adenoviruses is required for this interaction . These results suggest that interaction of CtBP with the E1A proteins may play a critical role in adenovirus replication and oncogenic transformation.

Oncogene, 1995 Nov 2, 11(9), 1675 - 83
Gadd45 is a nuclear cell cycle regulated protein which interacts with p21Cip1; Kearsey JM et al.; GADD45 was originally identified as a cDNA clone induced by growth arrest and DNA damage . We show that Gadd45 is a nuclear protein, widely expressed in normal tissues, particularly in quiescent cellular populations . Using cell synchronisation methods we show that Gadd45 levels are highest in the G1 phase of the cell cycle, and are greatly reduced during S phase . Immunoprecipitation of Gadd45 from mammalian cells reveals that it is tightly associated with a protein which reacts with antibodies to the cyclin dependent kinase inhibitor p21Cip1 . Binding of recombinant Gadd45 protein to overlapping p21Cip1 peptides in ELISA assays and use of the yeast two hybrid assay show that Gadd45 directly interacts with this cell cycle inhibitor . These data suggest that Gadd45 may act in the regulation of the cell cycle . It is postulated that the interactions of Gadd45 with both p21Cip1 and PCNA are important for the modulation of cell cycles, and for the inhibition of DNA replication.

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, 1995 Nov, 112(3), 285 - 9
Tributyltin affects phagocytic activity of Ciona intestinalis hemocytes; Cooper EL et al.; Organotin compounds have been used in marine anti-fouling paints as biocides . Because tunicates are vulnerable to these compounds in their natural habitats, we used Ciona intestinalis to establish an assay for phagocytosis in vitro of yeast by hemocytes after exposure to different concentrations (0.0015, 0.015, 0.15 and 1.5 microM) of four organotin compounds: tributyltin (TBT), triphenyltin (TPT), dibutyltin (DBT) and diphenyltin (DPT) . To evaluate the phagocytic activity, we used a method based on fluorescence excitation of yeast pre-treated with eosin-Y . The percentage of phagocytosis decreased from 45.1 +/- 3.49 to 22.4 +/- 5.14 at 1.5 microM of TBT (P < 0.001); it was significantly reduced in presence of the ionophore A23187 . TPT, DPT and DBT did not show significant effects on phagocytosis . Because the effect of TBT was irreversible, differences between the inhibitory mechanisms of ionophore and TBT are suggested . These results indicate that for future analyses, tunicates should become excellent models for dissecting events such as phagocytosis that are associated with immunosuppression after exposure to xenobiotics.

Protein Eng, 1995 Nov, 8(11), 1103 - 15
Use of a minimum perturbation approach to predict TIM mutant structures; Joseph-McCarthy D et al.; A minimum perturbation conformational search approach is used to model the structures of the yeast triosephosphate isomerase (TIM) single mutant in which the catalytic base Glu165 is changed to Asp, and the double mutant in which Glu165 is changed to Asp and Ser96 to Pro . In chicken TIM this double mutant is referred to as a pseudo-revertant because some of the catalytic activity lost due to the first mutation is regained when the second mutation occurs . Three minimum energy structures were calculated for the Asp165 conformation in the yeast TIM single mutant and another three for the double mutant . One of the calculated minimum energy conformations for Asp165 in the E165D structure agrees well with the X-ray structure . However, this conformation is not that of the lowest energy and is not one of the three most common conformers for Asp found by Ponder and Richards . This suggests that when an amino acid is introduced it may not be able to conform to the more general rules that apply to protein structures of evolutionary origin . While the van der Waals energy largely determines the allowed minima, the relative ranking of the final minima is determined by electrostatic effects and can therefore be affected by the inclusion of crystal waters in the calculation . When the E165D calculation is repeated with an active-site water molecule fixed in its E165D X-ray structure position, the relative ranking of the minima shifts and the X-ray conformation for Asp165 is the lowest interaction energy conformer . Two of the E165D calculated minimum energy structures are essentially identical to two of the S96P/E165D minima . All of the calculated minima for both the E165D and S96P/E165D mutants position the Asp side chain such that the anti-orbital, and not the more basic syn-orbital, of the carboxylate would be utilized for proton abstraction . This observation may explain why the chicken TIM S96P/E165D mutant, for which the X-ray structure indicates that the syn-orbital is used, is a pseudo-revertant while the yeast TIM double mutant is not; no X-ray structure is available for the latter . The multiplicity of minima found in the present analysis makes clear that predicting the exact orientation of a single side chain is not as simple as might be expected.

Genome Res, 1995 Nov, 5(4), 381 - 92
Generation of a high-resolution genetic map and a YAC contig of the Lurcher locus on mouse chromosome 6; Zuo J et al.; Lurcher (Lc) is a semidominant mouse mutant that displays progressive neurodegeneration during perinatal development . This genetic lesion results in apoptotic neuronal death in a dosage dependent and cell autonomous manner in specific neurons during their terminal differentiation . To understand the molecular basis of the Lc mutation, we have adopted a positional cloning approach based on its location on mouse chromosome 6 . To define the Lc locus, we have extended our previous analysis of an intersubspecific backcross between Mus m . castaneus and B6CBACa-Aw-j/A-Lc consisting of 504 animals (Norman et al . 1991) . In addition, 580 animals of a generic backcross between Mus spretus and C57BL/6 (The European Collaborative Interspecific Backcross) were utilized for the fine genetic mapping of the Lc locus . Using three RFLP markers and nine microsatellite markers in the vicinity of the Lc locus, we determined the order and relative genetic distances of these markers at a resolution of 0.1 cM . The Lc mutation was mapped between two flanking markers, D6Mit121 and D6Mit175, separated by a genetic distance of 0.5 cM . We then initiated the cloning of the genomic region surrounding these two markers by screening a YAC library and characterizing YAC end sequences for further screening . This effort has resulted in the construction of a YAC contig consisting of 14 YACs and spanning a 3-Mb region . Markers isolated from these YACs were used to further define the Lc locus, resulting in a physical map that places the Lc gene within an estimated 300-kb interval . This set of YACs and markers will serve as DNA sources for the identification of the Lc gene.

Genome Res, 1995 Nov, 5(4), 342 - 58
An integrated map of human chromosome 6p23; Olavesen MG et al.; The human chromosomal band 6p23 is a Giemsa-negative (light) band that may be expected to be relatively gene rich . The genes for spinocerebellar ataxia type 1 (SCA1), guanosine monophosphate reductase (GMPR), DEK involved in a subtype of acute myeloid leukemia (AML), and the folate-sensitive fragile site FRA6A, have already been mapped to 6p23 . Recent linkage data have suggested evidence for a susceptibility locus for schizophrenia in the region . We have constructed a single YAC contig of approximately 100 clones spanning the entire 6p23 band from 6p22.3 to 6p24.1 and covering 7.5-8.5 Mb of DNA . The YAC contig contains 55 markers including genetically mapped STSs, physically mapped STSs, anonymous STSs, anonymous ESTs, and ESTs from the genes mapped to the region . The order of the genetically mapped STSs is consistent with their order in the contig and some of the markers not resolved on the genetic map have been resolved by the YACs . Four of the YACs from 6p23 and covering approximately 3 Mb of DNA have been used to isolate approximately 300 cosmids from a flow-sorted human chromosome 6 cosmid library, which have been organized into pockets . The proposed susceptibility locus for schizophrenia is most closely linked to D6S260, which is located within the YAC contig along with genetic markers < or = 5 cM on either side . Therefore, the presented materials are valuable reagents for characterization of the genomic region implicated in schizophrenia.

Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1161 - 6
Fragmentation of the ribosome to investigate RNA-ligand interactions; Howard BA et al.; RNA molecules perform a variety of important and diverse functions and, therefore, an understanding of their structure and interaction with proteins and ligands is essential . Large RNA molecules (for example, the ribosomal RNAs) are complex and hence reports describing their fragmentation into functional subdomains has provided a means for their detailed analysis . We present here an in vivo approach to study RNA-ligand interactions . This is based on the concept that an RNA fragment could mimic a drug-binding site present on the intact molecule . Overexpression of the fragment would sequester the drug thereby permitting the continued functioning of the ribosome and, thus, ensuring cell viability . Accordingly, a fragment of 16S rRNA encompassing the spectinomycin-binding domain in helix 34 (nucleotides 1046-1065 and 1191-1211) was cloned and in vivo expression resulted in drug resistance . Furthermore, an RNA fragment lacking flanking sequences to helix 34 was also selected from among a pool of random rRNA fragments and shown to confer spectinomycin resistance . A similar in vitro approach is also described for the analysis of rRNA molecules that interact with the yeast elongation factor 3 (EF-3).

Mol Plant Microbe Interact, 1995 Nov-Dec, 8(6), 886 - 91
Genetic mapping of a wide spectrum nematode resistance gene (Hero) against Globodera rostochiensis in tomato; Ganal MW et al.; The Hero gene confers resistance to a wide spectrum of pathotypes of the potato cyst nematode Globodera rostochiensis . This gene has been introgressed from the wild tomato species Lycopersicon pimpinellifolium into the cultivated tomato . We have used RFLP and RAPD analysis for the targeted search of the L . pimpinellifolium into the cultivated tomato . We have used RFLP and RAPD analysis for the targeted search of the L . pimpinellifolium segment . The resistant line LA 1792 contains a single introgressed segment on chromosome 4, which is characterized by three RFLP markers from the high-density RFLP map of tomato . The map position of the Hero gene in large populations, four additional markers were identified in the introgressed region . After analyzing more than 800 gametes for recombination, we found that one marker is only 0.4 cM away from the Hero gene . YAC clones isolated from a region near the Hero gene indicate that in this area of the genome, the kb/cM ratio is relatively low (<450 kb/cM) and chromosome walking should be feasible in order to isolate this gene.

Scand J Clin Lab Invest, 1995 Nov, 55(7), 577 - 88
Studies of the release and turnover of a human neutrophil lipocalin; Axelsson L et al.; A 24-kDa protein was purified from human neutrophil extracts and shown to be the newly discovered neutrophil gelatinase-associated lipocalin (NGAL), based on structural and immunochemical data . A specific enzyme-linked immunosorbent assay (ELISA) was developed for the determination of NGAL in human plasma and tissue fluids . Normal human plasma contains 72 micrograms l-1 of NGAL (range 40-109 micrograms l-1) in two main forms, monomer and dimer . 35S-methionine metabolic studies of human neutrophils showed that granulocyte macrophagecolony-stimulating factor (GMCSF) stimulated significant synthesis and secretion of NGAL in a dose- and time-dependent fashion . NGAL was rapidly released as monomer and dimer on incubation of heparinized whole blood with opsonized yeast, reaching a plateau corresponding to about 35% of total cell content after 30 min . Following intravenous injection of 125-iodine labelled NGAL there was a more rapid initial clearance of the monomeric than of the dimeric form; t1/2 10 min vs . 20 min . During the second phase the two forms cleared at similar rates . Severe acute peritonitis was accompanied by a 10-fold increase in NGAL plasma levels and the NGAL level in peritoneal exudates, which reached about 40 mg l-1 . There was a good linear correlation between the concentrations of NGAL, leucocyte elastase and NP4 (neutrophil proteinase 4 = P3).

Somat Cell Mol Genet, 1995 Nov, 21(6), 415 - 28
Molecular analysis and breakpoint definition of a set of human chromosome 21 somatic cell hybrids; Graw SL et al.; Rodent-human somatic cell hybrids containing single human chromosomes or chromosome fragments are extremely valuable in physical mapping, marker analysis, and disease mapping . Chromosome 21 has been extensively studied in this fashion, and a single set of hybrids has been utilized in mapping the majority of chromosome 21 markers . The utility of a set of hybrids depends upon the definition of the human chromosome content . Recently, Chumakov and coworkers (1) utilized 198 chromosome 21 markers in the preliminary analysis of YACs spanning chromosome 21q . We have used these same markers to evaluate the STS content of a set of 27 chromosome 21 somatic cell hybrids, resulting in the description of the breakpoints at the molecular level, as well as the definition of 35 "bins . " The detailed molecular definition of chromosome 21 content of the hybrids, in combination with the further analysis of chromosome 21 YACs (2), has resulted in the most detailed picture of chromosome 21 to date.

Somat Cell Mol Genet, 1995 Nov, 21(6), 399 - 414
YAC analysis and minimal tiling path construction for chromosome 21q; Gardiner K et al.; We have undertaken a detailed analysis of several hundred YACs from widely available YAC libraries which map to human chromosome 21 with the goal of improving the physical map of chromosome 21 and determining the feasibility of producing a minimal tiling path of well characterized, stable, non-chimeric YACs spanning the long arm of the chromosome (21q) . We report information on over 500 YACs known to contain STS from 21q including information on size, stability, chimerism, marker content, and NotI restriction sites . YACs derive from the CEPH and St . Louis YAC libraries, and STSs include the set of 198 markers originally used do assemble a YAC contig of 21q, as well as additional anonymous probes and gene markers . This information has assisted in refinements of STS order, has defined a region of general instability in 2lq22.3, has identified an increased number of NotI restriction sites, and has defined cryptic gaps, particularly in 2lq2l, for which few or no markers are available . These results have allowed us to develop and assess a minimal tiling path of overlapping YACs consisting of 59 YACs (and two PI clones), largely non chimeric, stable, and of verified STS content . They total 30 mb of non-overlapping DNA, and contain all chromosome 21 specific STSs originally used to define the 810 YAC 21q YAC contig . When integrated with the analysis of a somatic cell hybrid mapping panel of chromosome 21 reported in the accompanying manuscript, a greatly enhanced understanding of the physical map of chromosome 21 is obtained.

Mamm Genome, 1995 Nov, 6(11), 802 - 4
Refined mapping of caltractin in human Xq28 and in the homologous region of the mouse X chromosome places the gene within the bare patches (Bpa) and striated (Str) critical regions; Chatterjee A et al.; Caltractin belongs to a family of calcium-binding proteins and is a structural component of the centrosome . A human caltractin cDNA (CALT) has recently been mapped by fluorescence in situ hybridization (FISH) to Xq28 . We report here refined mapping of the human CALT gene and its murine homolog between the loci DXS1104 (DXHXS1104) DXS52 (DXHXS52) by PCR and Southern analysis on YACs and somatic cell hybrids from the region in both species . These mapping studies place the gene within the critical region for the murine X-linked dominant, male lethal mutations bare batches and striated.

Genomics, 1995 Nov 1, 30(1), 89 - 90
The human immediate early gene BRF1 maps to chromosome 14q22-q24; Maclean KN et al.; BRF1 (Butyrate response factor 1) is a member of an immediate early gene family specifying putative nuclear transcription factors . A repeat motif incorporating two Cys and two His is highly conserved between family members identified from yeast, Drosophila, mouse, rat, and human . The chromosome localization of none of the human genes has been determined thus far . Using the polymerase chain reaction on a human-rodent hybrid panel, we have localized BRF1 to chromosome 14 . This was confirmed by direct sequencing of the PCR fragment . Using fluorescence in situ hybridization, the chromosome localization of BRF1 was further determined as 14q22-q24.

Genomics, 1995 Nov 1, 30(1), 81 - 3
Localization of the human achaete-scute homolog gene (ASCL1) distal to phenylalanine hydroxylase (PAH) and proximal to tumor rejection antigen (TRA1) on chromosome 12q22-q23; Renault B et al.; ASCL1, the human achaete-scute homolog, is a helix-loop-helix transcription factor that was previously assigned to chromosome 12 using a rodent-human somatic hybrid panel . We now placed this gene on a yeast artificial chromosome contig encompassing position 119 cM of the Genethon genetic map between the two genes phenylalanine hydroxylase (PAH) and tumor rejection antigen 1 (TRA1) . We also localized ASCL1 in the 12q22-q23 cytogenetic interval by using fluorescence in situ hybridization.

Genomics, 1995 Nov 1, 30(1), 37 - 45
An integrated YAC clone contig for the WAGR region on human chromosome 11p13-p14.1; Gawin B et al.; The WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) deletion region on chromosome 11p13 has been extensively characterized by deletion analysis and long-range restriction mapping . A dense probe set is available for this genomic region, which harbors a number of disease gene loci, some of which still are not cloned . The identification of candidates for these genes would be greatly facilitated by a complete gene map for this chromosomal segment . As an initial step toward this goal, we have isolated the entire region in 58 overlapping YAC clones . The contig spanning 8 Mb from RAG1 to KCNA4 has been assembled by STS and probe content mapping for 76 loci with an average spacing of about 100 kb . A subset of clones has been analyzed by PFG analysis to position these within the known physical map . Common microsatellite markers permit an alignment of the YAC contig with the genetic and radiation hybrid maps of chromosome 11 . Ten known genes, some with much more refined map positions, are placed in the contig . The severalfold coverage of 11p13-p14.1 provides a reliable resource for the future development of a complete gene map of this region.

Genomics, 1995 Nov 1, 30(1), 18 - 24
Human HOXB cluster and the nerve growth factor receptor gene: comparison with an orthologous chromosomal domain in mouse; Bentley KL et al.; The structural organization and nucleotide sequence similarity of mammalian Antennapedia-class homeobox genes support the view that the four homeobox clusters (HOXA, B, C, and D on human chromosomes 7, 17, 12, and 2, respectively) arose through a combination of gene duplication and divergence to form a cluster, followed by several cluster duplications . The duplication events that gave rise to the four clusters appear to have involved chromosomal domains extending well beyond the borders of the clusters in either direction . This evidence arises from the observation that many genes closely linked to the homeobox clusters on different chromosomes show sequence similarity . Here, we present a continuation of physical mapping studies to determine the extent and organization of the duplicated regions surrounding the four homeobox clusters in human . Southern blots prepared from pulsed-field gels of human DNA were probed with cloned segments of human HOXB genes and the nerve growth factor receptor (NGFR) gene on chromosome 17q21-q22 . Restriction enzyme analysis revealed the close physical linkage of these genes within 100 kb . Two yeast artificial chromosomes (YACs), 220 and 380 kb in size, were isolated using oligonucleotide primers specific for NGFR . Both YACs contained the entire HOXB cluster . Restriction mapping of the clones indicated that the distance separating these loci could not be greater than 50 kb . This result confirms and extends previous information on the proximity of these genes as determined by genetic linkage analysis and closely parallels the orthologous loci in the mouse.

Genomics, 1995 Nov 1, 30(1), 123 - 7
Localization of a human homolog of the mouse Tiam-1 gene to chromosome 21q22.1; Chen H et al.; Exon trapping was applied to genomic DNA from a chromosome 21-specific cosmid library (LL21NC02-Q) to clone portions of genes from this chromosome . Among a large number of trapped exons, three showed striking homology to different regions of the cDNA for the mouse T-lymphoma invasion and metastasis gene (Tiam-1) at both nucleotide and predicted amino acid sequence levels, suggesting that these three exons are part of a human homolog of the mouse Tiam-1 gene . We mapped this presumed human TIAM1 gene to chromosome 21 by using appropriate somatic cell hybrids, YACs, and cosmids . The TIAM1 gene localizes to YAC 760H5 of the I . Chumakov et al . (1992, Nature 359: 380) YAC contig between markers D21S298 and D21S404 in band 21q22.1 . This human gene (which is a member of the group of guanine nucleotide-dissociation stimulators that modulate the activity of Rho-like proteins) may be important in the development or metastasis of malignancies that are associated with abnormalities on chromosome 21, including the various forms of leukemia frequent in trisomy 21.

Br J Pharmacol, 1995 Nov, 116(6), 2625 - 30
Competitive inhibition of coumarin 7-hydroxylation by pilocarpine and its interaction with mouse CYP 2A5 and human CYP 2A6; Kinonen T et al.; 1 . We have shown earlier that pilocarpine strongly inhibits mouse and human liver coumarin 7-hydroxylase activity of CYP 2A and pentoxyresorufin O-deethylase activity of CYP 2B in vitro . Since pilocarpine, like coumarin, contains a lactone structure we have studied in more detail its inhibitory potency on mouse and human liver coumarin 7-hydroxylation . 2 . Pilocarpine was a competitive inhibitor of coumarin 7-hydroxylase in vitro both in mouse and human liver microsomes although it was not a substrate for CYP 2A5 . Ki values were similar, 0.52 +/- 0.22 microM in mice and 1.21 +/- 0.51 microM in human liver microsomes . 3 . Pilocarpine induced a type II difference spectrum in mouse, human and recombinant CYP 2A5 yeast cell microsomes, with Ka values of 3.7 +/- 1.6, 1.6 +/- 1.1 and 1.5 +/- 0.1 microM, respectively . 4 . Increase in pH of the incubation medium from pH 6 to 7.5 increased the potency of inhibition of coumarin 7-hydroxylation by pilocarpine . 5 . Superimposition of pilocarpine and coumarin in such a way that their carbonyls, ring oxygens and the H-7' of coumarin and N-3 of pilocarpine overlap yielded a common molecular volume of 82% . 6 . The results indicate that pilocarpine is a competitive inhibitor and has a high affinity for mouse CYP 2A5 and human CYP 2A6 . In addition the immunotype nitrogen of pilocarpine is coordinated towards the haem iron in these P450s.

Hum Mol Genet, 1995 Nov, 4(11), 2047 - 55
Detailed mapping and loss of heterozygosity analysis suggests a suppressor locus involved in sporadic breast cancer within a distal region of chromosome band 17p13.3; Stack M et al.; The chromosome region 17p13.3 is thought to encode a tumour suppressor gene involved in sporadic breast cancer and other malignancies . Physical ordering of markers has been carried out by a series of multicolour fluorescent in situ hybridisation (FISH) experiments, using isolated yeast artificial chromosomes (YACs) and cosmids . Eight polymorphic markers ordered within this new physical map and one external marker were used to investigate the pattern of loss of heterozygosity in a panel of 40 sporadic breast tumour patients . The data revealed a region of high loss (60%) within distal 17p13.3, defined by markers D17S926, D17S695 and D17S849 which mapped close together . A contig of YACs was constructed physically linking these three markers.

Hum Mol Genet, 1995 Nov, 4(11), 2025 - 32
The complete sequence of the coding region of the ATM gene reveals similarity to cell cycle regulators in different species; Savitsky K et al.; Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency radiation sensitivity, and cancer predisposition . A-T heterozygotes are moderately cancer prone . The A-T gene, designated ATM, was recently identified in our laboratory by positional cloning, and a partial cDNA clone was found to encode a polypeptide with a PI-3 kinase domain . We report here the molecular cloning of a cDNA contig spanning the complete open reading frame of the ATM gene . The predicted protein of 3056 amino acids shows significant sequence similarities to several large proteins in yeast, Drosophila and mammals, all of which share the PI-3 kinase domain . Many of these proteins are involved in the detection of DNA damage and the control of cell cycle progression . Mutations in their genes confer a variety of phenotypes with features similar to those observed in human A-T cells . The complete sequence of the ATM gene product provides useful clues to the function of this protein, and furthers understanding of the pleiotropic nature of the A-T mutations.

Genes Chromosomes Cancer, 1995 Nov, 14(3), 204 - 9
Partial chromosome 21 amplification in a child with acute lymphoblastic leukemia; Le Coniat M et al.; Monosomy 21 and metacentric markers corresponding in size to chromosomes 8 to 12 were found as the only clonal chromosomal changes in a child with acute lymphoblastic leukemia (ALL) . chromosome painting with a whole chromosome 21-specific probe showed that the marker originated from chromosome 21 . Fluorescence in situ hybridization with yeast artificial chromosome (YAC) probes to chromosome 21 showed genomic amplification with two, four, or more copies of the probed DNA sequences present on the marker . The most amplified regions of chromosome 21 were centromeric and telomeric to the Down's syndrome region . This observation supports the notion that amplification of only parts of chromosome 21 may be important in the leukemogenic process in spite of the high incidence of complete trisomy 21 in ALL.

Genetics, 1995 Nov, 141(3), 903 - 7
Evolutionary origin of nonuniversal CUGSer codon in some Candida species as inferred from a molecular phylogeny; Pesole G et al.; CUG, a universal leucine codon, has been reported to be read as serine in various yeast species belonging to the genus Candida . To gain a deeper insight into the origin of this deviation from the universal genetic code, we carried out a phylogenetic analysis based on the small-subunit ribosomal RNA genes from some Candida and other related Hemiascomycetes . Furthermore, we determined the phylogenetic relationships between the tRNA(Ser)CAG, responsible for the translation of CUG, from some Candida species and the other serine and leucine isoacceptor tRNAs in C . cylindracea . We demonstrate that the group of Candida showing the genetic code deviation is monophyletic and that this deviation could have originated more than 150 million years ago . We also describe how phylogenetic analysis can be used for genetic code predictions.

J Am Med Inform Assoc, 1995 Nov-Dec, 2(6), 351 - 64
Internet-based support for bioscience research: a collaborative genome center for human chromosome 12; Miller PL et al.; This paper describes an approach that provides Internet-based support for a genome center to map human chromosome 12, as a collaboration between laboratories at the Albert Einstein College of Medicine in Bronx, New York, and the Yale University School of Medicine in New Haven, Connecticut . Informatics is well established as an important enabling technology within the genome mapping community . The goal of this paper is to use the chromosome 12 project as a case study to introduce a medical informatics audience to certain issues involved in genome informatics and in the Internet-based support of collaborative bioscience research . Central to the approach described is a shared database (DB/12) with Macintosh clients in the participating laboratories running the 4th Dimension database program as a user-friendly front end, and a Sun SPARCstation-2 server running Sybase . The central component of the database stores information about yeast artificial chromosomes (YACs), each containing a segment of human DNA from chromosome 12 to which genome markers have been mapped, such that an overlapping set of YACs (called a "contig") can be identified, along with an ordering of the markers . The approach also includes 1) a map assembly tool developed to help biologists interpret their data, proposing a ranked set of candidate maps, 2) the integration of DB/12 with external databases and tools, and 3) the dissemination of the results . This paper discusses several of the lessons learned that apply to many other areas of bioscience, and the potential role for the field of medical informatics in helping to provide such support.

Acta Paediatr, 1995 Nov, 84(11), 1241 - 4
Fruit juice malabsorption: not only fructose; Hoekstra JH et al.; Malabsorption of free fructose, when ingested in excess over glucose, is considered a significant factor in apple juice induced diarrhoea . Absorption of the carbohydrates in fruit juices was investigated by means of the hydrogen breath test in 15 healthy children aged 2.2-6.4 years, consuming 15 ml kg-1 of each juice with a maximum of 375 ml . Incomplete absorption was found following the ingestion of apple juice (5/5), grape juice (10/10) and bilberry juice (8/10), although the last two contain equivalent concentrations of fructose and glucose . When the same tests were repeated after yeast treatment of the juices, which leads to major reductions in fructose and glucose contents, malabsorption was found to persist . No symptoms were observed following any of the tests . Our results suggest a significant role for other carbohydrates than fructose, possibly those originating from the fruit skin, with respect to fruit juice-induced breath hydrogen excretion.

J Cell Biochem, 1995 Nov, 59(3), 389 - 401
Arrest at the G2/M transition of the cell cycle by protein-tyrosine phosphatase inhibition: studies on a neuronal and a glial cell line; Faure R et al.; The addition of the peroxovanadium (pV) derivatives potassium bisperoxo(1,10-phenanthroline)oxovanadate(v) (bpV{phen}) or potassium bisperoxo(pyridine-2-carboxylato) oxovanadate(v) (bpV{pic}), both of which are potent inhibitors of protein tyrosine phosphatases (PTPs) {Posner et al . (1994): J Biol Chem 269:4596-4604}, to the culture medium of neuroblastoma NB 41 and glioma C6 cells resulted in a marked decrease in their proliferation rates and a progressive accumulation at the G2/M transition of the cell cycle . The effect was dependent on dose, cell type, and a pV compound employed . Mean values of the RNA-to-DNA and RNA-to-protein ratios in NB cells treated for 48 h with increased doses of bpV{phen} showed that general synthetic functions were not altered, nor did we observe oxidative damage to DNA using a sensitive DNA-nick detection assay . No changes in the expression and localization of vimentin, a component of the intermediate filament cytoskeleton, were observed by indirect immunofluorescence, showing that treatment did not disturb the cytoskeleton network . Measurements of BrdU incorporation into newly synthesized DNA showed that cells treated were not totally arrested . Furthermore, cells arrested G2/M were able to reenter the cycle rapidly after the release of inhibition . This progressive accumulation of G2/M coincided with the detection of tyrosine-phosphorylated p34cdc2 and a dramatic reduction in its kinase activity toward histone H1 by 48 h of culture . Both compounds were equally potent in inhibiting the catalytic activity of a yeast and the structurally distant mouse cdc25B in vitro, suggesting that augmented tyrosine phosphorylation of p34cdc2 derived from the in vivo inhibition of cdc25 . Their equal in vitro potency contrasted with the considerably greater potency of bpV{phen} in vivo, in vivo suggesting that factors regulating the intracellular access of these compounds to cdc25 might be critical in determining in vivo specificity . In conclusion the final consequence of long-term exposure to potent and structurally defined PTP inhibitors on two highly proliferative nerve cell lines is to restrict cell growth . The corresponding hyperphosphorylation and reduced activity of p34cdc2 likely reflects the unusual sensitivity of cdc25 as an in vivo target for peroxovanadium compounds.

Cell Growth Differ, 1995 Nov, 6(11), 1395 - 403
Normal p53 status and function despite the development of drug resistance in human breast cancer cells; Wosikowski K et al.; Loss of or mutations in p53 protein have been shown to decrease both radio- and chemosensitivity . The present study assessed the p53 gene status, ability to arrest in G1 of the cell cycle, the functionality of the p53 transduction pathway, and apoptosis following treatment with radiation in a series of drug-resistant human breast cancer cells to determine whether p53 alterations occur during the development of drug resistance . We used 13 sublines derived from MCF-7, ZR75B, and T47D cells, which were resistant to doxorubicin, paclitaxel, vinblastine, cisplatin, etoposide, and amsacrine . Eleven of 12 drug-resistant sublines retained the parental p53 gene status, as determined by sequence analysis and functional yeast assay; only one subline was found to have acquired a mutation in the p53 gene . The MCF-7 TH subline was found to both acquire mutated p53 and to have major changes in p53 protein expression and function . In 12 other drug-resistant sublines, the G1 checkpoint was conserved or only slightly impaired . A normal accumulation of p53, p21Cip1/Waf1, and Mdm2 proteins and hypophosphorylation of Rb protein occurred in response to radiation with only small differences noted in the kinetics of p53 and p21Cip1/Waf1 induction . Increased susceptibility to apoptosis was found in the ZR75B drug-resistant sublines, whereas no evidence for apoptosis was observed in the ZR75B, MCF-7, and T47D parentals and the MCF-7 and T47D drug-resistant sublines . This effect could not be explained by alterations in bcl-2 or bax expression . Our results demonstrate that alterations in: (a) p53 gene status; (b) ability to arrest in G1; (c) induction of p53 protein and p53-dependent genes; and (d) decreased activation of apoptosis is not a requirement for the onset of drug resistance . The function of p53 appears to be dissociated from drug resistance in our model system.

Plant Mol Biol, 1995 Nov, 29(4), 627 - 35
Senescence-induced expression of a homologue of delta 9 desaturase in rose petals; Fukuchi-Mizutani M et al.; cDNAs for senescence-inducible genes were isolated by differential hybridization from a cDNA library derived from mRNAs from the petals of rose flowers . The amino acid sequence deduced from these cDNAs exhibited significant homology to those of delta 9 acyl-lipid desaturases of cyanobacteria and of delta 9 acyl-CoA desaturases of a yeast and mammals . There was no amino-terminal sequence indicative of a leader peptide for targeting to the chloroplasts or to mitochondria . Northern blot analysis indicated that the transcripts of the cDNAs were expressed specifically in petals at late developmental stages and during senescence . It is proposed that a delta 9 desaturase in the senescing petals play an important role in the degradation of saturated fatty acids of membrane lipids.

Plant Cell, 1995 Nov, 7(11), 1773 - 85
Ectopic expression of the Arabidopsis transcriptional activator Athb-1 alters leaf cell fate in tobacco; Aoyama T et al.; The Arabidopsis thaliana Athb-1 is a homeobox gene of unknown function . By analogy with homeobox genes of other organisms, its gene product, Athb-1, is most likely a transcription factor involved in developmental processes . We constructed a series of Athb-1-derived genes to examine the roles of Athb-1 in transcriptional regulation and plant development . Athb-1 was found to transactivate a promoter linked to a specific DNA binding site by transient expression assays . In transgenic tobacco plants, overexpression of Athb-1 or its chimeric derivatives with heterologous transactivating domains of the yeast transcription factor GAL4 or herpes simplex virus transcription factor VP16 conferred deetiolated phenotypes in the dark, including cotyledon expansion, true leaf development, and an inhibition of hypocotyl elongation . Expression of Athb-1 or the two chimeric derivatives also affected the development of palisade parenchyma under normal growth conditions, resulting in light green sectors in leaves and cotyledons, whereas other organs in the transgenic plants remained normal . Both developmental phenotypes were induced by glucocorticoid in transgenic plants expressing a chimeric transcription factor comprising the Athb-1 DNA binding domain, the VP16 transactivating domain, and the glucocorticoid receptor domain . Plants with severe inducible phenotypes showed additional abnormality in cotyledon expansion . Our results suggest that Athb-1 is a transcription activator involved in leaf development.

Plant J, 1995 Nov, 8(5), 763 - 70
The CIC library: a large insert YAC library for genome mapping in Arabidopsis thaliana; Creusot F et al.; A new Arabidopsis thaliana (ecotype Columbia) genomic library has been constructed in Yeast Artificial Chromosomes: the CIC library (for CEPH, INRA and CNRS) . Optimization of plant culture conditions and protoplast preparation allowed the recovery of large amounts of viable protoplasts . Mechanical shearing of DNA was minimized by isolation of DNA from protoplasts embedded in agarose . Cloning of large inserts was favored by including two successive size fractionation steps (after partial EcoRI digestion and after ligation with the vector arms), which selected DNA fragments larger than 350 kb . The library consists of 1152 clones with an average insert size of 420 kb . Clones carrying chloroplast DNA and various nuclear repeated sequences have been identified . Twenty-one per cent of the clones are found to contain chloroplast DNA . Therefore, the library represents around four nuclear genome equivalents . The clones containing 5S rDNA genes, 18S-25S rDNA sequences and the 180 bp paracentromeric repeated element account for 3.6%, 8.9% and 5.8%, respectively . Only one clone was found to carry the 160 bp paracentromeric repeated element . Given the smaller size of clones carrying Arabidopsis repeated DNA, the average size of remaining clones is around 480 kb . The library was screened by PCR amplification using pairs of primers corresponding to sequences dispersed in the genome . Seventy out of 76 pairs of primers identified from one to seven YAC clones . Thus at least 92% of the genome is represented in the CIC library . The survey of the library for clones containing unlinked DNA sequences indicates that the proportion of chimeric clones is lower than 10%.

Eur J Biochem, 1995 Nov 1, 233(3), 880 - 5
Affinity labeling of recombinant ricin A chain with Procion blue MX-R; Alderton WK et al.; Recombinant ricin A chain was irreversibly modified by Procion blue MX-R, a dichlorotriazinyl analogue of Cibacron blue F3G-A, at pH 7.5 and 4 degrees C in 90 h with over 95% loss of activity in an in vitro translation assay . The presence of total yeast RNA reduced the covalent attachment of Procion blue MX-R to ricin A chain . Quantitatively modified ricin A chain contained 2 mol Procion blue MX-R/mol 29-kDa subunit . Tryptic digestion and resolution of the peptides by reverse-phase high-performance liquid chromatography yields a blue peptide corresponding to Gln5-Arg26 of ricin A chain . Thus, a likely dye-binding site on recombinant ricin A was identified . This region is removed from the active-site cleft of recombinant ricin A but may be involved in its substrate binding.

J Neurochem, 1995 Nov, 65(5), 1955 - 66
A new Cys2/His2 zinc finger gene, rKr2, is expressed in differentiated rat oligodendrocytes and encodes a protein with a functional repressor domain; Pott U et al.; The function of the vertebrate nervous system is dependent on the proper myelination of its fiber tracts . Myelin of the CNS is produced by oligodendrocytes . To identify gene regulatory proteins expressed in this particular glial cell type, we isolated cDNAs coding for Cys2/His2 zinc finger proteins from a rat oligodendrocyte cDNA library . One clone, named rKr2 (rKr for rat Kruppel-type protein), encodes a protein with 19 carboxy-terminal zinc finger domains and an amino-terminal Kruppel-associated box domain . This amino-terminal domain of the rKr2 protein behaved as a strong transcriptional repressor module when fused to the DNA binding domain of yeast GAL4 and tested on an appropriate reporter construct . High levels of rKr2 mRNA in adult rat tissues were found only in the CNS and testis; in the CNS, the message was predominantly expressed in differentiated oligodendrocytes . The modular structure of the rKr2 protein (carboxy-terminal DNA binding domain, amino-terminal repressor module) and its expression pattern suggest that it acts as a sequence-specific transcriptional repressor in the myelin-producing glial cells of the CNS.

J Cell Biol, 1995 Nov, 131(3), 807 - 16
Beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin beta 3 subunit; Shattil SJ et al.; The adhesive and signaling functions of integrins are regulated through their cytoplasmic domains . We identified a novel 111 residue polypeptide, designated beta 3-endonexin, that interacted with the cytoplasmic tail of the beta 3 integrin subunit in a yeast two-hybrid system . This interaction is structurally specific, since it was reduced by 64% by a point mutation in the beta 3 cytoplasmic tail (S752-->P) that disrupts integrin signaling . Moreover, this interaction is integrin subunit specific since it was not observed with the cytoplasmic tails of the alpha IIb, beta 1, or beta 2 subunits . beta 3-Endonexin fusion proteins bound selectively to detergent-solubilized beta 3 from platelets and human umbilical vein endothelial cells, and beta 3-endonexin mRNA and protein were detected in platelets and other tissues . A related mRNA encoded a larger polypeptide that failed to bind to beta integrin tails . The apparent specificity of beta 3-endonexin for the beta 3 integrin subunit suggests potential mechanisms for selective modulation of integrin functions.

J Cell Biol, 1995 Nov, 131(3), 619 - 30
Targeting signals and subunit interactions in coated vesicle adaptor complexes; Page LJ et al.; There are two clathrin-coated vesicle adaptor complexes in the cell, one associated with the plasma membrane and one associated with the TGN . The subunit composition of the plasma membrane adaptor complex is alpha-adaptin, beta-adaptin, AP50, and AP17; while that of the TGN adaptor complex is gamma-adaptin, beta'-adaptin, AP47, and AP19 . To search for adaptor targeting signals, we have constructed chimeras between alpha-adaptin and gamma-adaptin within their NH2-terminal domains . We have identified stretches of sequence in the two proteins between amino acids approximately 130 and 330-350 that are essential for targeting . Immunoprecipitation reveals that this region determines whether a construct coassemblies with AP50 and AP17, or with AP47 and AP19 . These observations suggest that these other subunits may play an important role in targeting . In contrast, beta- and beta'-adaptins are clearly not involved in this event . Chimeras between the alpha- and gamma-adaptin COOH-terminal domains reveal the presence of a second targeting signal . We have further investigated the interactions between the adaptor subunits using the yeast two-hybrid system . Interactions can be detected between the beta/beta'-adaptins and the alpha/gamma-adaptins, between the beta/beta'-adaptins and the AP50/AP47 subunits, between alpha-adaptin and AP17, and between gamma-adaptin and AP19 . These results indicate that the adaptor subunits act in concert to target the complex to the appropriate membrane.

Genes Dev, 1995 Nov 1, 9(21), 2672 - 83
The 160-kD subunit of human cleavage-polyadenylation specificity factor coordinates pre-mRNA 3'-end formation; Murthy KG et al.; Cleavage-polyadenylation specificity factor (CPSF) is a multisubunit protein that plays a central role in 3' processing of mammalian pre-mRNAs . CPSF recognizes the AAUAAA signal in the pre-mRNA and interacts with other proteins to facilitate both RNA cleavage and poly(A) synthesis . Here we describe the isolation of cDNAs encoding the largest subunit of CPSF (160K) as well as characterization of the protein product . Antibodies raised against the recombinant protein inhibit polyadenylation in vitro, which can be restored by purified CPSF . Extending previous studies, which suggested that 160K contacts the pre-mRNA, we show that purified recombinant 160K can, by itself, bind preferentially to AAUAAA-containing RNAs . While the sequence of 160K reveals similarities to the RNP1 and RNP2 motifs found in many RNA-binding proteins, no clear match to a known RNA-binding domain was found, and RNA recognition is therefore likely mediated by a highly diverged or novel structure . We also show that 160K binds specifically to both the 77K (suppressor of forked) subunit of the cleavage factor CstF and to poly(A) polymerase (PAP) . These results provide explanations for previously observed cooperative interactions between CPSF and CstF, which are responsible for poly(A) site specification, and between CPSF and PAP, which are necessary for synthesis of the poly(A) tail . Also supporting a direct role for 160K in these interactions is the fact that 160K by itself retains partial ability to cooperate with CstF in binding pre-mRNA and, unexpectedly, inhibits PAP activity in in vitro assays . We discuss the significance of these multiple functions and also a possible evolutionary link between yeast and mammalian polyadenylation suggested by the properties and sequence of 160K.

Genes Dev, 1995 Nov 1, 9(21), 2569 - 82
Abi-2, a novel SH3-containing protein interacts with the c-Abl tyrosine kinase and modulates c-Abl transforming activity; Dai Z et al.; A protein has been identified that interacts specifically with both the Src homologous 3 (SH3) domain and carboxy-terminal sequences of the c-Abl tyrosine kinase . The cDNA encoding the Abl interactor protein (Abi-2), was isolated from a human lymphocyte library using the yeast two-hybrid system with the Abl SH3 domain as bait . Abi-2 binds to c-Abl in vitro and in vivo . Abi-2 is a novel protein that contains an SH3 domain and proline-rich sequences critical for binding to c-Abl . A basic region in the amino terminus of Abi-2 is homologous to the DNA-binding sequence of homeo-domain proteins . We show that Abi-2 is a substrate for the c-Abl tyrosine kinase . Expression of an Abi-2 mutant protein that lacks sequences required for binding to the Abl SH3 domain but retains binding to the Abl carboxyl terminus activates the transforming capacity of c-Abl . The properties of Abi-2 are consistent with a dual role as regulator and potential effector of the c-Abl protein and suggest that Abi-2 may function as a tumor suppressor in mammalian cells.

Blood, 1995 Nov 1, 86(9), 3404 - 12
Inhibition of erythro-myeloid differentiation by constitutive expression of a DNA binding-deficient c-myb mutant: implication for c-myb function; Sala A et al.; The c-myb proto-oncogene encodes a nuclear protein involved in the regulation of cell proliferation, differentiation, and development . Myb protein contains a DNA binding and a transactivating domain thought to mediate its biologic properties . The DNA binding domain consists of three repeats (R1, R2, and R3), each containing a highly conserved motif of tryptophan residues . A c-myb mutant (DR1-myb) lacking the last 46 amino acids of R1 and 23 amino terminal residues of R2, a region homologous to the ADA-2 yeast transcriptional adaptor, lost DNA binding ability, but remained able to transactivate the human heat-shock promoter . Transfection of murine 32D and murine erythroleukemia (MEL) cell lines with DR1-myb caused inhibition of cellular differentiation induced by granulocyte colony-stimulating factor (G-CSF) and dimethyl sulfoxide (DMSO), respectively . A second c-myb mutant (D-ADA2-myb) lacking the first 23 amino acids of R2, also lost DNA binding and transactivation activity, but did not inhibit DMSO-induced differentiation of MEL transfected cells . These findings suggest that deletion of R1 activates a DNA binding-independent mechanism of c-myb function, which may involve interaction of Myb with cellular factors.

Mol Cell Biol, 1995 Nov, 15(11), 5937 - 44
Requirement of the SH3 and SH2 domains for the inhibitory function of tyrosine protein kinase p50csk in T lymphocytes; Cloutier JF et al.; Previous studies from our laboratory have shown that the cytosolic tyrosine protein kinase p50csk is involved in the negative regulation of T-cell activation (L.M . L . Chow, M . Fournel, D . Davidson, and A . Veillette, Nature {London} 365:156-160, 1993) . This function most probably reflects the ability of Csk to phosphorylate the inhibitory carboxy-terminal tyrosine of p56lck and p59fynT, two Src-related enzymes abundantly expressed in T lymphocytes . Herein, we have attempted to better understand the mechanisms by which Csk participates in the inhibitory phase of T-cell receptor signalling . Our results demonstrated that the Src homology 3 (SH3) and SH2 domains of p50csk are crucial for its negative impact on T-cell receptor-mediated signals . As these two sequences were not essential for phosphorylation of the carboxy-terminal tyrosine of a Src-like product in yeast cells, we postulated that they mediate protein-protein interactions allowing the recruitment of p50csk in the vicinity of activated Lck and/or FynT in T cells . In complementary studies, it was observed that linkage of a constitutive membrane targeting signal to the amino terminus of Csk rescued the deleterious impact of a point mutation in the SH2 domain of p50csk . This observation suggested that the SH2 sequence is in part necessary to translocate p50csk from the cytoplasm to the plasma membrane, where Src-related enzymes are located . Nevertheless, constitutive membrane localization was unable to correct the effect of complete deletion of the SH3 or SH2 sequence, implying that these domains provide additional functions necessary for the biological activity of p50csk.

J Mol Evol, 1995 Nov, 41(5), 563 - 72
Fungal origin by horizontal transfer of a plant mitochondrial group I intron in the chimeric CoxI gene of Peperomia; Vaughn JC et al.; We present phylogenetic evidence that a group I intron in an angiosperm mitochondrial gene arose recently by horizontal transfer from a fungal donor species . A 1,716-bp fragment of the mitochondrial coxI gene from the angiosperm Peperomia polybotrya was amplified via the polymerase chain reaction and sequenced . Comparison to other coxI genes revealed a 966-bp group I intron, which, based on homology with the related yeast coxI intron aI4, potentially encodes a 279-amino-acid site-specific DNA endonuclease . This intron, which is believed to function as a ribozyme during its own splicing, is not present in any of 19 coxI genes examined from other diverse vascular plant species . Phylogenetic analysis of intron origin was carried out using three different tree-generating algorithms, and on a variety of nucleotide and amino acid data sets from the intron and its flanking exon sequences . These analyses show that the Peperomia coxI gene intron and exon sequences are of fundamentally different evolutionary origin . The Peperomia intron is more closely related to several fungal mitochondrial introns, two of which are located at identical positions in coxI, than to identically located coxI introns from the land plant Marchantia and the green alga Prototheca . Conversely, the exon sequence of this gene is, as expected, most closely related to other angiosperm coxI genes . These results, together with evidence suggestive of co-conversion of exonic markers immediately flanking the intron insertion site, lead us to conclude that the Peperomia coxI intron probably arose by horizontal transfer from a fungal donor, using the double-strand-break repair pathway . The donor species may have been one of the symbiotic mycorrhizal fungi that live in close obligate association with most plants.

EMBO J, 1995 Nov 1, 14(21), 5387 - 98
dpa, a member of the MCM family, is required for mitotic DNA replication but not endoreplication in Drosophila; Feger G et al.; We have isolated the Drosophila disc proliferation abnormal (dpa) gene, a member of the MCM family of DNA replication factors . Members of this family of proteins are required for DNA replication in yeast . A dpa null mutant dies during pupal stages because imaginal tissues necessary for the formation of the adult fly fail to proliferate normally . Beginning in late embryogenesis BrdU labeling reveals DNA replication defects in mitotically proliferating cells . In contrast, dpa is dispensable for endoreplication, a specialized cell cycle consisting of consecutive rounds of S phases without intervening mitosis . Our studies suggest an essential role for dpa in mitotic DNA replication but not in endoreplication . Thus, dpa is not a general replication factor but may play a specialized regulatory role in DNA replication.

EMBO J, 1995 Nov 1, 14(21), 5279 - 87
The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain; Pearson RB et al.; The immunosuppressive agent rapamycin induces inactivation of p70s6k with no effect on other mitogen-activated kinases . Here we have employed a combination of techniques, including mass spectrometry, to demonstrate that this effect is associated with selective dephosphorylation of three previously unidentified p70s6k phosphorylation sites: T229, T389 and S404 . T229 resides at a conserved position in the catalytic domain, whose phosphorylation is essential for the activation of other mitogen-induced kinases . However, the principal target of rapamycin-induced p70s6k inactivation is T389, which is located in an unusual hydrophobic sequence outside the catalytic domain . Mutation of T389 to alanine ablates kinase activity, whereas mutation to glutamic acid confers constitutive kinase activity and rapamycin resistance . The importance of this site and its surrounding motif to kinase function is emphasized by its presence in a large number of protein kinases of the second messenger family and its conservation in putative p70s6k homologues from as distantly related organisms as yeast and plants.

J Biol Chem, 1995 Oct 27, 270(43), 25898 - 904
Initiation of Xenopus oocyte maturation by activation of the mitogen-activated protein kinase cascade; Gotoh Y et al.; Mitogen-activated protein kinase (MAPK) and MAPK kinase (MAPKK) are activated during Xenopus oocyte maturation concomitant with the activation of maturation promoting factor (MPF) . We reported previously that an anti-MAPKK neutralizing antibody inhibited progesterone- or Mos- induced initiation of oocyte maturation . Here, we show that the injection of CL100 (also called MAPK phosphatase-1) into immature oocytes inhibited progesterone-induced oocyte maturation as well as MAPK activation and that injection of mRNA encoding a constitutively active MAPKK induced activation of histone H1 kinase and germinal vesicle breakdown in the absence of progesterone . Injection of recombinant STE11 protein (a yeast MAPKK kinase) also induced initiation of oocyte maturation . These data support the idea that the MAPKK/MAPK cascade plays an important role in oocyte maturation . Interestingly, injection of the active MAPKK mRNA or the STE11 protein resulted in induction and accumulation of Mos protein . Furthermore, in the presence of cycloheximide, the STE11-induced activation of MPF as well as the induction and accumulation of Mos was blocked, and the activation of MAPK was greatly reduced . The increase in Mos protein and the activation of MAPK by injecting cyclin A protein into immature oocytes were both blocked also by cycloheximide treatment . These results are consistent with an idea that there may exist a positive feedback loop consisting of Mos, the MAPKK/MAPK cascade, and MPF, which may be important for the initiation of oocyte maturation induced by progesterone.

Nucleic Acids Res, 1995 Oct 25, 23(20), 4127 - 33
Isolation of YAC insert sequences by representational difference analysis; Schutte M et al.; We present a method for the isolation of YAC insert sequences by representational difference analysis (RDA) . To achieve maximal representation of the sequences, the amplicons were generated from a Mbol digestion product . RDA was performed using a 970 kb insert YAC clone . After two rounds of re-association and selective amplification 92% of the difference product represented sequences derived from the YAC insert . Twenty insert-specific sequence-tagged sites were readily defined . The difference product was also successfully used to isolate microsatellite markers, to identify clones from a human PAC library and as a chromosome painting probe in fluorescence in situ hybridization.

Biochemistry, 1995 Oct 24, 34(42), 13943 - 8
Sequential domain unfolding in phosphoglycerate kinase: fluorescence intensity and anisotropy stopped-flow kinetics of several tryptophan mutants; Beechem JM et al.; Stopped-flow total intensity and anisotropy experiments on single tryptophan containing mutants of yeast phosphoglycerate kinase (PGK) located in either the carboxy-terminal domain (W308 and W333), amino-terminal domain (W48 and W122), or "hinge" region (W194 and W399) were performed . The results obtained for single tryptophans in individual domains suggest that the unfolding of PGK by guanidinium hydrochloride is a sequential process in which unfolding of the carboxy-terminal domain is followed by the unfolding of the amino-terminal domain . A kinetic intermediate has been detected which consists of an unfolded carboxy-terminal domain and an altered amino-terminal domain, identical in hydrodynamic properties with the native state, but hyperfluorescent . In contrast to the C-terminal tryptophans, which exhibit concurrent total intensity and anisotropy changes in the entire denaturant concentration range (0-->2 M), the N-terminal tryptophans experience a large increase in fluorescence intensity and a constant anisotropic environment at low concentrations of denaturant, corresponding to the first transition region of the equilibrium unfolding profile . Anisotropy changes for the N-terminal probes are observed above 1 M Gdn-HCl, the region corresponding to the second equilibrium unfolding transition . Stopped-flow experiments performed on PGK mutants with two tryptophans (i.e., with a single tryptophan in each domain) confirm that each domain unfolds independently, and that the individual site-specific mutations do not significantly alter the unfolding pathway . Unfolding kinetics experiments with tryptophans situated in the hinge reveal that the region sensed by W399 unfolds before the carboxy-terminal domain, whereas W194 senses unfolding of both domains.

Biochem Biophys Res Commun, 1995 Oct 24, 215(3), 987 - 93
DNA end-joining in extracts from human cells; Boe SO et al.; DNA end-joining is a central feature of several DNA recombination processes . A DNA end-joining activity present in extracts prepared from cells of the human SupT1 lymphocyte cell line was characterised . Joining of blunt ends and ends having complementary single-strand extensions (SSEs) were precise with no insertion or deletion of substrate base pairs . DNA sequencing analysis showed that molecules having non complementary ends of the same polarity, or molecules having one blunt end and one end with a SSE, were joined without loss of nucleotide sequences in the double-stranded region of the substrate molecule . The joining patterns observed have several features that are consistent with DNA end-joining activities previously observed in vitro in extracts from Xenopus eggs and in vivo in mammalian cells and yeast.

Proc Natl Acad Sci U S A, 1995 Oct 24, 92(22), 10287 - 91
Grb-IR: a SH2-domain-containing protein that binds to the insulin receptor and inhibits its function; Liu F et al.; To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries . A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR . The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR . To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR . Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR . Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated . The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1 . The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited . These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

Science, 1995 Oct 20, 270(5235), 480 - 3
Physical map and organization of Arabidopsis thaliana chromosome 4; Schmidt R et al.; A physical map of Arabidopsis thaliana chromosome 4 was constructed in yeast artificial chromosome clones and used to analyze the organization of the chromosome . Mapping of the nucleolar organizing region and the centromere integrated the physical and cytogenetic maps . Detailed comparison of physical with genetic distances showed that the frequency of recombination varied substantially, with relative hot and cold spots occurring along the whole chromosome . Eight repeated DNA sequence families were found in a complex arrangement across the centromeric region and nowhere else on the chromosome.

J Biol Chem, 1995 Oct 20, 270(42), 24761 - 8
Assembly of voltage-gated potassium channels . Conserved hydrophilic motifs determine subfamily-specific interactions between the alpha-subunits; Xu J et al.; Voltage-gated potassium (K+) channels are assembled by four identical or homologous alpha-subunits to form a tetrameric complex with a central conduction pore for potassium ions . Most of the cloned genes for the alpha-subunits are classified into four subfamilies: Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw), and Kv4 (Shal) . Subfamily-specific assembly of heteromeric K+ channel complexes has been observed in vitro and in vivo, which contributes to the diversity of K+ currents . However, the molecular codes that mediate the subfamily-specific association remain unknown . To understand the molecular basis of the subfamily-specific assembly, we tested the protein-protein interactions of different regions of alpha-subunits . We report here that the cytoplasmic NH2-terminal domains of Kv1, Kv2, Kv3, and Kv4 subfamilies each associate to form homomultimers . Using the yeast two-hybrid system and eight K+ channel genes, two genes (one isolated from rat and one from Drosophila) from each subfamily, we demonstrated that the associations to form heteromultimers by the NH2-terminal domains are strictly subfamily-specific . These subfamily-specific associations suggest a molecular basis for the selective formation of heteromultimeric channels in vivo.

Nature, 1995 Oct 19, 377(6550), 649 - 52
Crystal structure of double-stranded DNA containing the major adduct of the anticancer drug cisplatin; Takahara PM et al.; The success of cisplatin in cancer chemotherapy derives from its ability to crosslink DNA and alter the structure . Most cisplatin-DNA adducts are intrastrand d(GpG) and d(ApG) crosslinks, which unwind and bend the duplex to facilitate the binding of proteins that contain one or more high-mobility group (HMG) domains . When HMG-domain proteins such as HMG1, IXR (intrastrand-crosslink recognition) protein from yeast, or human upstream-binding factor (hUBF) bind cisplatin intrastrand crosslinks, they can be diverted from their natural binding sites on the genome and shield the adducts from excision repair . These activities sensitize cells to cisplatin and contribute to its cytotoxic properties . Crystallographic information about the structure of cisplatin-DNA adducts has been limited to short single-stranded deoxyoligonucleotides such as cis-{Pt(NH3)2(d(pGpG))} . Here we describe the X-ray structure at 2.6 A resolution of a double-stranded DNA dodecamer containing this adduct . Our information provides, to our knowledge, the first crystallographic look at a platinated DNA duplex and should help the design of new platinum and other metal crosslinking antitumour drug candidates . Moreover, the structure reveals a unique fusion of A- and B-type DNA segments that could be of more general importance.

Genes Dev, 1995 Oct 15, 9(20), 2495 - 508
Homeless is required for RNA localization in Drosophila oogenesis and encodes a new member of the DE-H family of RNA-dependent ATPases; Gillespie DE et al.; The homeless (hls) gene of Drosophila is required for anteroposterior and dorsoventral axis formation during oogenesis . At a low frequency, females homozygous for mutations in hls generate early egg chambers in which the oocyte is positioned incorrectly within the cyst . At a high frequency, late-stage egg chambers exhibit a ventralized chorion . Sequence analysis of the hls cDNA predicts a protein with amino-terminal homology to members of the DE-H family of RNA-dependent ATPases and putative helicases . Similarity of 51% in the amino-terminal third of the protein was found to two yeast splicing factors, PRP2 and PRP16, and to Drosophila Maleless, which is required for dosage compensation . To analyze Hls function, RNA localization patterns were determined for seven different transcripts in hls mutant ovaries . Previtellogenic transport to the oocyte was unaffected for all transcripts examined . Transport and localization of bicoid and oskar messages during vitellogenic stages were strongly disrupted, and the distribution and/or quantity of gurken, orb, and fs(1)K10 mRNAs were also affected, but to a lesser degree . In contrast, hu-li tai shao and Bicaudal-D transcripts were transported and localized normally in hls mutants . In addition, Kinesin heavy chain:beta-Galactosidase fusion protein failed to localize correctly to the posterior of the oocyte in vitellogenic egg chambers . Examination of the microtubule structure with anti-alpha-Tubulin antibodies revealed aberrant microtubule organizing center movement and an abnormally dense cytoplasmic microtubule meshwork . We discuss potential roles for Hls in organizing a cytoskeletal framework essential for localizing specific RNAs.

Blood, 1995 Oct 15, 86(8), 3118 - 22
Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals; Biernaux C et al.; The major bcr-abl fusion gene is presently seen as the hallmark of chronic myeloid leukemia (CML) and presumably as the cause of its development . Accordingly, long-term disappearance of bcr-abl after intensive therapy is considered to be a probable cure of CML . The nested reverse transcriptase-polymerase chain reaction (RT-PCR) provides a powerful tool for minimal residual CML detection . The RT-PCR was optimized by (1) increasing the amount of total RNA involved in the reverse transcription reaction to correspond to total RNA extracted from 10(8) cells, (2) using a specific abl primer in this reverse reaction, and (3) reamplifying 10% of the RT-PCR product in nested amplification . This optimized RT-PCR permitted us to detect up to 1 copy of RNA bcr-abl synthesised in vitro, mixed with yeast RNA in an equivalent quantity to 10(8) white blood cells (WBCs) . Using this highly sensitive RT-PCR during the follow-up of CML patients, a signal was unexpectedly found in healthy controls . Therefore, a systematic study of the possible expression of bcr-abl RNA in the WBCs of healthy adults and children and in umbilical cord blood was undertaken . It showed the presence of bcr-abl transcript in the blood of 22 of 73 healthy adults and in the blood of 1 of 22 children but not in 22 samples of umbilical cord blood.

Cancer Res, 1995 Oct 15, 55(20), 4640 - 5
Microdissection based cloning of a translocation breakpoint in a human malignant melanoma; Zhang J et al.; Chromosome translocations in human malignancies have identified the genomic location of several important growth-regulatory sequences (e.g., cellular oncogenes and suppressor genes) . Melanomas are characterized by recurring chromosome alterations, including deletion or translocations of the long arm of chromosome 6 (6q) . This report details our efforts to clone the t(1;6)(q21;q14) breakpoint in a malignant melanoma to further our understanding of the biology of these tumors . The strategy utilized combined microdissection of the translocation chromosome, development and characterization of a DNA microclone library, isolation of cosmids and YACs from the breakpoint region, ordering of clones by two-color metaphase/interphase fluorescence in situ hybridization, and finally, identification of a YAC spanning the translocation breakpoint . By analogy to other tumor systems, molecular examination of the chromosome 6 breakpoint may provide insight into the pathobiology of this important neoplasm.

Cancer Res, 1995 Oct 15, 55(20), 4570 - 4
An integrated high-resolution physical map of the DPC/BRCA2 region at chromosome 13q12; Schutte M et al.; We identified a homozygous deletion in a pancreatic carcinoma (DPC) that localized to a 1-cM region at chromosome 13q12.3, which lay within the 6-cM locus of familial breast cancer susceptibility (BRCA-2) . Here we present a physical map of the region, consisting of YAC, PAC, and cosmid contigs . The YAC contig comprises 16 clones that together span the entire BRCA2 region . The PAC contig comprises 22 clones that together span the DPC region . Seventy cosmid clones were localized within and near the DPC region . Thirty-five sequence-tagged sites were defined and localized within the map . The map indicates the size of the DPC region to be near 250 kb, and provides mapped and cloned resources for the search for the putative tumor suppressor gene(s) in the region.

Neurosci Lett, 1995 Oct 13, 199(1), 73 - 7
Mutation analysis of the chromosome 14q24.3 dihydrolipoyl succinyltransferase (DLST) gene in patients with early-onset Alzheimer disease; Cruts M et al.; Linkage analysis studies have indicated that the chromosome band 14q24.3 harbours a major gene for familial early-onset Alzheimer's disease (AD) . Recently we localized the chromosome 14 AD gene (AD3) in the 6.4 cM interval between the markers D14S289 and D14S61 . We mapped the gene encoding dihydrolipoyl succinyltransferase (DLST), the E2k component of human alpha-ketoglutarate dehydrogenase complex (KGDHC), in the AD3 candidate region using yeast artificial chromosomes (YACs) . The DLST gene is a candidate for the AD3 gene since deficiencies in KGDHC activity have been observed in brain tissue and fibroblasts of AD patients . The 15 exons and the promoter region of the DLST gene were analysed for mutations in chromosome 14 linked AD cases and in two series of unrelated early-onset AD cases (onset age < 55 years) . Sequence variations in intronic sequences (introns 3, 5 and 10) or silent mutations in exonic sequences (exons 8 and 14) were identified . However, no AD related mutations were observed, suggesting that the DLST gene is not the chromosome 14 AD3 gene.

Nature, 1995 Oct 12, 377(6549), 548 - 52
A WD-domain protein that is associated with and phosphorylated by the type II TGF-beta receptor; Chen RH et al.; Transforming growth factor-beta (TGF-beta) is the prototype for a family of extracellular polypeptides that affect cell proliferation and differentiation, and tissue morphogenesis . TGF-beta signalling is mediated by two types of serine/threonine kinase receptors, the type I and II receptors, which are able to form a heteromeric complex . No cytoplasmic proteins that associate with these receptors in vivo, or are their kinase targets, have yet been described . We have now identified a WD-domain-containing protein, TRIP-1, which specifically associates with the type II TGF-beta receptor in a kinase-dependent way . TRIP-1 does not interact with the type II activin or type I receptors, but associates with the heteromeric TGF-beta receptor complex . TRIP-1 is phosphorylated on serine and threonine by the receptor kinase, strongly suggesting that it has a role in TGF-beta signalling . This is supported by coexpression of TRIP-1 and type II receptor during development . The existence of TRIP-1 homologues in plant and yeast suggests a conserved function in all eukaryotes.

Nucleic Acids Res, 1995 Oct 11, 23(19), 3842 - 9
Fine-mapping of shotgun template-libraries; an efficient strategy for the systematic sequencing of genomic DNA; Scholler P et al.; To test the effectiveness of ordering shotgun DNA-templates prior to sequence analysis, the 450 kb left arm of yeast chromosome XII was randomly subcloned into a phagemid vector . Clones were ordered by hybridisation to an average map density of one new insert every 125 bp and are currently used for sequencing the chromosomal fragment . An 11.5 kb overlap between the template map and a DNA fragment that had been sequenced earlier allowed an independent evaluation of the strategy's effectiveness . To this end, clones were selected from the map and tag-sequenced from either end, thus comparing the map position with the actual location within the 11.5 kb . Of 65 selected clones, taken mostly at random from a total of 423, 58 mapped on average about a quarter of a clone length around their predicted position, with the other seven being between 0.6 and 1.5 clone length off . 75-86 sequencing reactions on clones selected from the map would have been sufficient for completely sequencing both strands of the 11.5 kb fragment . The results demonstrate the efficacy of such template sorting, considerably assisting sequencing at relatively little cost on the mapping level.

Genomics, 1995 Oct 10, 29(3), 796 - 800
Expression analysis, genomic structure, and mapping to 7q31 of the human sperm adhesion molecule gene SPAM1; Jones MH et al.; During the course of systematic sequence tag analysis of clones isolated from an adult testis cDNA library, clones 296 and 576 were found to detect 71-74% sequence identity to the guinea pig sperm surface protein PH-20 . This surface protein is involved in sperm-egg adhesion in the guinea pig . Nucleotide sequence for 1919 bp of human DNA from a series of overlapping cDNA clones isolated from a testis cDNA library confirmed the sequence identity within a 1527-bp open reading frame to be 71-74% to the guinea pig gene and the similarity to be 60% for the predicted protein of 509 amino acids . Southern blot analysis of human genomic DNA and DNA from somatic cell hybrids indicates that the gene (SPAM1) is unique and does not form part of a larger family and that it maps to chromosome 7 . Fluorescence in situ hybridization with yeast artificial chromosome (YAC) clones isolated from the CEPH megaYAC library has refined this localization to 7q31 . PCR analysis of genomic DNA and YAC clone DNA has shown that the 1919 bp of the gene that has been cloned covers approximately 11 kb of genomic DNA and is encoded by at least 4 exons . Northern analysis of poly(A)+ mRNA from a range of 16 human tissues has demonstrated that expression of the gene as a single 2.4-kb transcript is strictly limited to the testis.

Genomics, 1995 Oct 10, 29(3), 787 - 92
Characterization of a YAC contig spanning the pseudoautosomal region; Ried K et al.; Due to its unique biology of partial sex linkage and high recombination rates, the pseudoautosomal region (PAR1) on both X and Y chromosomes has attracted considerable interest . In addition, an extremely high level of YAC instability has been observed in this region . We have derived 82 YAC clones from six different YAC libraries mapping to this 2.6-Mb region . Of these a subset of 22 YACs was analyzed in detail . YAC contigs were assembled using 67 pseudoautosomal probes, of which 64 were unambiguously ordered . All markers are well distributed over the entire region, including the middle part of the region, which has previously been found difficult to contig . Two gaps of less than 50 kb within the genomic locus of CSF2RA and around XE7 remain, which could not be covered with YACs, cosmids, or phages . This YAC contig anchored on the physical map of PAR1 represents one of the best characterized large regions of the human genome with a map completion greater than 90% at 100-kb resolution and has permitted the accurate localization of all known genes within this region.

Genomics, 1995 Oct 10, 29(3), 766 - 8
Physical mapping of the human ELA1 gene between D12S361 and D12S347 on chromosome 12q13; Davies RL et al.; ELA1, the pancreatic elastase 1 gene, is conserved in mammalian genomes . ELA1 was previously mapped to chromosome 12 using a panel of mouse-human somatic cell hybrids . We now report the physical and cytogenetic localization of the ELA1 gene . On the physical map, ELA1 is adjacent to the polymorphic marker AFMa283yg1 and between D12S361 and D12S347 . Using fluorescence in situ hybridization, we determined that ELA1 maps to 12q13.

Genomics, 1995 Oct 10, 29(3), 725 - 31
The melanoma antigen gene (MAGE) family is clustered in the chromosomal band Xq28; Rogner UC et al.; The melanoma antigen gene (MAGE) family comprises 12 known genes, of which 6 are expressed in tumors . In the course of a systematic analysis of transcripts in Xq28, we have identified cDNAs related to different MAGE genes . Analysis of cell hybrids, ordered YACs, and cosmids showed that all MAGE genes are located in Xq28 and are clustered in three main intervals within 3.5 Mb . The six genes expressed in tumors are contained in the two intervals closest to the telomere and are highly homologous to each other . Analysis of different species suggests that human MAGE sequences are conserved in primates, but less well conserved in other vertebrate species.

Genomics, 1995 Oct 10, 29(3), 647 - 52
Physical linkage of the human growth hormone gene cluster and the skeletal muscle sodium channel alpha-subunit gene (SCN4A) on chromosome 17; Bennani-Baiti IM et al.; The human growth hormone (GH) locus, a cluster of five genes, spans 47 kb on chromosome 17q22-q24 . The skeletal muscle sodium channel alpha-subunit locus (SCN4A), a 32.5-kb gene, has previously been mapped to 17q23.1-q25.3 . We demonstrate that both the GH gene cluster and the SCN4A gene colocalize to a single 525-kb yeast artificial chromosome (YAC) containing DNA derived from human chromosome 17 . Restriction maps of two cosmids encompassing the 5' terminus of the GH locus and including up to 40 kb of 5'-flanking sequences demonstrate a perfect 20-kb overlap with previously published maps of the SCN4A gene . A 720-bp DNA segment, encompassing sequences 32.3 to 31.6 kb 5' to GH, was sequenced and found to be identical to exon 14 of SCN4A . These data demonstrate that the SCN4A gene and the entire GH gene cluster are contained within 100 kb on chromosome 17 and are separated by only 21.5 kb . Remarkably, this physical linkage between GH and SCN4A also reveals that multiple elements critical to tissue-specific transcriptional activation of the GH gene lie within the SCN4A gene.

Proc Natl Acad Sci U S A, 1995 Oct 10, 92(21), 9638 - 42
Identification of a 95-kDa WEE1-like tyrosine kinase in HeLa cells; Parker LL et al.; Human WEE1 (WEE1Hu) was cloned on the basis of its ability to rescue wee1+ mutants in fission yeast {Igarashi, M., Nagata, A., Jinno, S., Suto, K . & Okayama, H . (1991) Nature (London) 353, 80-83} . Biochemical studies carried out in vitro with recombinant protein demonstrated that WEE1Hu encodes a tyrosine kinase of approximately 49 kDa that phosphorylates p34cdc2 on Tyr-15 {Parker, L . L . & Piwnica-Worms, H . (1992) Science 257, 1955-1957} . To study the regulation of WEE1Hu in human cells, two polyclonal antibodies to bacterially produced p49WEE1Hu were generated . In addition, a peptide antibody generated against amino acids 361-388 of p49WEE1Hu was also used . Unexpectantly, these antibodies recognized a protein with an apparent molecular mass of 95 kDa in HeLa cells, rather than one of 49 kDa . Immunoprecipitates of p95 phosphorylated p34cdc2 on Tyr-15, indicating that p95 is functionally related to p49WEEIHu, and mapping studies demonstrated that p95 is structurally related to p49WEE1Hu . In addition, the substrate specificity of p95 was more similar to that of fission yeast p107wee1 than to that of human p49WEE1 . Finally, the kinase activity of p95 toward p34cdc2/cyclin B was severely impaired during mitosis . Taken together, these results indicate that the original WEE1Hu clone isolated in genetic screens encodes only the catalytic domain of human WEE1 and that the authentic human WEE1 protein has an apparent molecular mass of approximately 95 kDa.

Proc Natl Acad Sci U S A, 1995 Oct 10, 92(21), 9530 - 4
A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution; Wong WT et al.; In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain) . This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heterogeneous proteins of yeast and nematode . The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation . We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic origin . These observations prompted our search for additional EH-containing proteins in mammalian cells . Using an EH domain-specific probe derived from the eps15 cDNA, we cloned and characterized a cDNA encoding an EH-containing protein with overall similarity to Eps15; we designated this protein Eps15r (for Eps15-related) . Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins.

Proc Natl Acad Sci U S A, 1995 Oct 10, 92(21), 9525 - 9
A nuclear hormone receptor-associated protein that inhibits transactivation by the thyroid hormone and retinoic acid receptors; Burris TP et al.; Nuclear hormone receptors are transcription factors that require multiple protein-protein interactions to regulate the expression of their target genes . Using the yeast two-hybrid system, we identified a protein, thyroid hormone receptor uncoupling protein (TRUP), that specifically interacts with a region of the human thyroid hormone receptor (TR) consisting of the hinge region and the N-terminal portion of the ligand binding domain in a hormone-independent manner . Interestingly, TRUP inhibits transactivation by TR and the retinoic acid receptor but has no effect on the estrogen receptor or the retinoid X receptor in mammalian cells . We also demonstrate that TRUP exerts its action on TR and retinoic acid receptor by interfering with their abilities to interact with their DNA . TRUP represents a type of regulatory protein that modulates the transcriptional activity of a subclass of the nuclear hormone receptor superfamily by preventing interaction with their genomic response elements.

Proc Natl Acad Sci U S A, 1995 Oct 10, 92(21), 9445 - 9
Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo; Cosentino GP et al.; The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro . A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize . In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization . In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA . These results suggest that the binding of dsRNA by PKR is necessary for dimerization . The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay . Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo . Such complexes could mediate differential effects on gene expression and control of cell growth.

Cell, 1995 Oct 6, 83(1), 101 - 10
The C . elegans gene lin-44, which controls the polarity of certain asymmetric cell divisions, encodes a Wnt protein and acts cell nonautonomously; Herman MA et al.; Mutations in the C . elegans gene lin-44 lead to reversals in the polarity of certain asymmetric cell divisions . We have discovered that lin-44 is a member of the Wnt family of genes, which encode secretory glycoproteins implicated in intercellular signaling . Both in situ hybridization experiments using lin-44 transcripts and experiments using reporter constructs designed to mimic patterns of lin-44 expression indicate that lin-44 is expressed in hypodermal cells at the tip of the tail and posterior to the cells with polarities affected by lin-44 mutations . Our mosaic analysis indicates that lin-44 acts cell nonautonomously . We propose that LIN-44 protein is secreted by tail hypodermal cells and affects the polarity of asymmetric cell divisions that occur more anteriorly in the tail.

Nature, 1995 Oct 5, 377(6548), 397 - 404
Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor; Horlein AJ et al.; Thyroid-hormone and retinoic-acid receptors exert their regulatory functions by acting as both activators and repressors of gene expression . A nuclear receptor co-repressor (N-CoR) of relative molecular mass 270K has been identified which mediates ligand-independent inhibition of gene transcription by these receptors, suggesting that the molecular mechanisms of repression by thyroid-hormone and retinoic-acid receptors are analogous to the co-repressor-dependent transcriptional inhibitory mechanisms of yeast and Drosophila.

Oncogene, 1995 Oct 5, 11(7), 1283 - 90
A single ancestral gene of the human LIM domain oncogene family LMO in Drosophila: characterization of the Drosophila Dlmo gene; Zhu TH et al.; Members of the human TTG/RBTN family, now renamed 'LMO' for LIM-only proteins, encode proteins with two tandem copies of a LIM motif . There are three members of this family, two have been isolated at the sites of chromosomal translocations in T-cell leukaemia . The function of the LIM motifs is at present unknown . We found that the LMO-2 gene is highly conserved between mammals, Drosophila and yeast . As a first step to obtain a model system for studying the function of the LIM motifs we have isolated the Drosophila homologue Dlmo . In contrast to mammals Drosophila appears to have only one lmo gene . A 2087 bp cDNA clone was isolated from a larval cDNA library, encoding a protein of 266 amino acids . A second transcript with an alternative 5' end was identified in RNA from embryos . The Drosophila lmo protein consists of two tandem copies of the conserved LIM domain characteristic of the human LMO family and an extended amino and carboxy terminus, which is not present in the human proteins . The amino acid sequence similarity with human LMO-1 and LMO-2 in LIM 1 is 79% and 69% and in LIM-2 90% and 60%, respectively . In addition a short stretch of 25 nucleotides with a homology of 83% between LMO-2 and Dlmo is found in the 3' UTR . Dlmo, like LMO-1, has an intron after the second LIM encoding region, which is not present in LMO-2 . It is expressed maternally and at a high level in early embryogenesis as well as in adults . Interestingly we observed that the Dlmo protein is immunologically related to LMO-2 and can be detected by immunohistochemistry in early cellular blastoderm embryos . The gene was localised to a genetically well characterized region (17C on the X chromosome) opening the way for identification of mutations.

Gene, 1995 Oct 3, 163(2), 327 - 8
Cloning and characterization of a cDNA encoding the specific phosphatidylcholine transfer protein from bovine liver; Cohen DE et al.; A 1917-bp cDNA clone encoding phosphatidylcholine transfer protein (PC-TP) was identified by screening a bovine liver library . Northern blot analysis demonstrated a 2-kb mRNA transcript in bovine liver, and Southern blotting was consistent with a single bovine PC-TP gene which was shown to be present in a diverse group of vertebrates, but not in yeast . Database comparisons revealed the nucleotide sequence of the clone to be unique and unrelated to other cytosolic lipid TP.

Gene, 1995 Oct 3, 163(2), 243 - 7
Rat intestinal crypt-cell replication factor with homology to early S-phase proteins required for cell division; Sykes DE et al.; Cell proliferation requires inhibitory and permissive factors to monitor cell-cycle progression and control DNA replication . The small intestine has a high rate of proliferation and a very low incidence of cancer, suggestive of efficient mechanisms for control of the cell cycle and assuring fidelity of DNA replication . We have isolated a cDNA from a rat crypt-cell library which hybridized to a 3.0-kb mRNA specific for crypt cells, the proliferative cell compartment of the intestine . Its amino-acid sequence indicates that it is a new member of a family of replication proteins found in yeast, Cenorhabditis elegans, mouse and humans . Its transcripts were markedly increased in fetal rat intestine and liver, decreased in long-term confluent and serum-starved tissue culture cells (IEC cells, a cell line derived from rat crypt cells), increased with serum repletion as cells resumed proliferation, and appeared to be species specific . Isolation and functional characterization of small intestinal crypt-cell replication factors should help explain this organ's low incidence of cancer.

Biochemistry, 1995 Oct 3, 34(39), 12892 - 902
Prolyl isomerase as a probe of stability of slow-folding intermediates; Veeraraghavan S et al.; Catalysis of slow folding reactions by peptidyl prolyl cis-trans isomerase (PPI) provides estimates of stabilities of intermediates in folding of normal and mutational variants of yeast iso-2 cytochrome c . A two-state model postulating a rapid preequilibration of intermediates with the unfolded protein is employed to calculate the stabilization free energy of the intermediate from the catalytic efficiency (kcat/Km) of PPI toward slow folding species . Stability measurements have been made for two distinct slow-folding intermediates: the absorbance-detected (IIS) and fluorescence-detected (IIIS) intermediates . Mutation-induced changes in the stability of the intermediates and in the activation free energy for slow folding are compared to changes in equilibrium thermodynamic stability . The results show that (1) for iso-2 the absorbance-detected intermediates (IIS) are slightly more stable than the fluorescence-detected intermediates (IIIS), (2) most mutations have different effects on equilibrium stability and the stability of the IIS or IIIS intermediates, and (3) for both slow folding reactions the mutation-induced changes in the activation free energy are small compared to the magnitude of the activation free energy barrier . Differential effects of mutations on equilibrium stability and the stability of intermediates provides a means of assessing the sequence-encoded structural specificity for folding . Mutations with different effects on intermediate stability and equilibrium stability change the encoded folding information and may alter folding pathways and/or lead to different three-dimensional structures . Identification of mutations which stabilize a folding intermediate relative to the native conformation provides an empirical approach to the design of thermodynamically stable forms of folding intermediates.

Biochemistry, 1995 Oct 3, 34(39), 12812 - 9
Probing the folding mechanism of a leucine zipper peptide by stopped-flow circular dichroism spectroscopy; Zitzewitz JA et al.; Leucine zipper peptides provide simple model systems for studying both the intramolecular and intermolecular interactions that govern protein folding . The synthetic 33-residue peptide GCN4-p1, derived from the yeast transcriptional activator GCN4, forms a stable biomolecular coiled-coil structure {O'Shea, E . K., Klemm, J . D., Kim, P . S., & Alber, T . (1991) Science 254, 539-544} . The guanidine-HCl induced equilibrium unfolding of this peptide at 5 degrees C and pH 7.0 yields a standard state free energy of 10.49 +/- 0.23 kcal (mol dimer)-1 when fit to a two-state model involving the native dimer and the unfolded monomer . The unfolding and refolding kinetics of GCN4-p1 were monitored by stopped-flow circular dichroism spectroscopy as a function of both peptide concentration and final denaturant concentration . The unfolding kinetics displayed single-exponential behavior, consistent with a unimolecular reaction . The refolding kinetics, which are dependent on both peptide and guanidine concentration, are well described by a simple bimolecular association reaction . A simultaneous fit of all of the unfolding and refolding kinetic data to the model, N2{symbol: see text}2U, yields refolding and unfolding rate constants in the absence of denaturant of 4.2 x 10(5) M-1 S-1 and 3.3 x 10(-3) S-1, respectively . The equilibrium unfolding curve is accurately predicted from these rate constants, providing further support for the validity of the two-state kinetic model.

Biochemistry, 1995 Oct 3, 34(39), 12513 - 23
X-ray structure and catalytic mechanism of lobster enolase; Duquerroy S et al.; Enolase prepared from lobster tail muscle yielded trigonal crystals with one 47 kDa subunit per asymmetric unit . X-ray data were collected on the apoenzyme at 2.4 A resolution and on a complex with Mn2+ and the inhibitor phosphoglycolate at 2.2 A resolution . The corresponding cDNA was amplified from a library of lobster muscle cDNA, and a sequence corresponding to residues 27-398 was determined . It is highly homologous to other enolases, including yeast enolase for which an X-ray structure is available . Yeast enolase was used as a starting point for crystallographic refinement, which led to models of lobster enolase having R-factors below 22% and good stereochemistry . These models are very similar to yeast enolase; they have the same fold with a beta 3 alpha 4 N-terminal domain followed by an atypical alpha/beta barrel . Lobster apoenolase and the ternary complex differ only in the position of three mobile loops . In the complex, a single Mn2+ ion is seen ligated to three carboxylates and three water molecules . Phosphoglycolate binds near, but not directly to, the metal . His 157, which belongs to one of the mobile loops, is in contact with the C2 atom of the ligand . A water molecule hydrogen-bonds to the carboxylate of the ligand and to those of Glu 166 and Glu 209 . We suggest that His 157 is the base that abstracts the C2H proton, whereas the water molecule is part of a proton relay system keeping the substrate in the carboxylic acid form where the pKa of the C2H group is low enough for proton transfer to His 157 . The resulting catalytic mechanism is different from those proposed on the basis of the yeast enzyme X-ray structures, but it fits with earlier biochemical and spectroscopic data.

Neurochem Int, 1995 Oct-Nov, 27(4-5), 417 - 24
Effect of ammonia on endocytosis and cytokine production by immortalized human microglia and astroglia cells; Atanassov CL et al.; Ammonium acetate decreased in a concentration-dependent manner the phagocytic uptake of mannosylated latex microspheres and of yeast by immortalized human microglia (CHME-5) and astroglioma (GL-15) cells . In both cell lines ammonium acetate affected also the secretion of certain cytokines . The most conspicuous effects were the following: in both cell lines ammonium acetate enhanced greatly the secretion of tumor necrosis factor-alpha in the absence of any other stimulus . in the human microglia cells ammonia decreased the constitutive secretion of interleukin-6, but it enhanced the stimulated (interleukin-1 alpha, tumor necrosis factor-alpha, gamma-interferon and gamma-interferon + tumor necrosis factor-alpha) secretion of interleukin-8 . In the astroglioma cell line, the stimulated release of tumor necrosis factor-alpha, interleukin-6 and interleukin-8 was diminished by ammonium acetate . The magnitude of the ammonia-effect depended on the stimulating agent (lipopolysaccharide, interleukin-1 alpha, tumor necrosis factor-alpha, gamma-interferon) . The results are discussed with regard to their potential importance in the pathogenesis of human diseases with elevated blood and brain ammonia concentrations.

Clin Nucl Med, 1995 Oct, 20(10), 909 - 12
Three-phase bone and Ga-67 scintigraphy in disseminated sporotrichosis; Patange V et al.; A 27-year-old man, who had been shoveling gravel in southern Texas for 3 years, had a history of papules and nodules in the left lateral wall of the abdomen . The lesions increased in number and severity with spread to other regions of the body . A punch biopsy of the right arm lesion revealed intracellular, round and cigar shaped budding yeast . The cultures grew Sporotrichum schenkii . Three-phase bone imaging and a Ga-67 scan defined the extent of the disease including involvement of the right tibia, left second metacarpal, and the left wrist joints, the latter two of which were not apparent on clinical examination.

Hum Mol Genet, 1995 Oct, 4(10), 1935 - 44
A new human gene from the Down syndrome critical region encodes a proline-rich protein highly expressed in fetal brain and heart; Fuentes JJ et al.; Down syndrome is a major cause of mental retardation and congenital heart defects . While most of the affected individuals have three copies of chromosome 21, patients with partial trisomy 21 have also been described . These rare cases define a minimal region for the Down syndrome phenotype encompassing about 3 Mb around D21S55 . By using a new method for the identification of coding sequences (Alu-splice PCR) we have identified a new gene, DSCR1, from region 21q22.1-q22.2 . DSCR1 encodes a novel protein which has an acidic domain, a serine-proline motif, a putative DNA binding domain and a proline-rich region with the characteristics of a SH3 domain ligand . These features suggest that DSCR1 could be involved in transcriptional regulation and/or signal transduction . DSCR1 is highly expressed in human brain and heart, and increased expression in the brains of young rats compared with adults suggests a role for DSCR1 during central nervous system development . Structural characteristics, together with its particular expression in brain and heart, encourage us to suggest that the overexpression of DSCR1 may be involved in the pathogenesis of Down syndrome, in particular mental retardation and/or cardiac defects.

Hum Mol Genet, 1995 Oct, 4(10), 1903 - 10
Quantitative DNA fiber mapping; Weier HU et al.; The assembly of sequence ready, high-resolution physical maps and construction of minimally overlapping contigs for the human as well as model genomes requires accurate determination of the extent of overlap between adjacent clones as well as their relative orientation . This is presently done by procedures such as clone fingerprinting, Southern blot analysis or clone end sequencing . We present a complementary analytical technique to map directly cloned DNA sequences on to individual stretched DNA molecules . This approach uses the hydrodynamic force of a receding meniscus to prepare straight high molecular weight DNA molecules that provide a linear template of approximately 2.3 kb/microns on to which the cloned probes can be mapped by in situ hybridization . This technique has numerous advantages such as a very high density of mapping templates, reproducible stretching of the mapping template providing a linear genomic scale, determination of clone orientation and direct visualization of DNA repeats . The utility and accuracy of quantitative DNA fiber mapping are illustrated through three examples: (i) mapping of lambda DNA restriction fragments along linearized approximately 49 kb long lambda phage DNA molecules with approximately 1 kb precision; (ii) localization of the overlap between a cosmid and a colinear P1 clone; and (iii) mapping of P1 clones along an approximately 490 kb yeast artificial chromosome (YAC) with approximately 5 kb precision and estimation of the approximately 25 kb gap between them.

Hum Mol Genet, 1995 Oct, 4(10), 1821 - 7
An integrated physical and genetic map of a 35 Mb region on chromosome Xp22.3-Xp21.3; Ferrero GB et al.; We have constructed a detailed physical map of the 35 Mb region spanning human chromosome Xp22.3-Xp21.3 . The backbone of the map is represented by a single oriented contiguous stretch of 585 overlapping yeast artificial chromosome (YAC) clones covering the entire region . The map is formatted with 615 map objects that include 324 YACs, 185 sequence tagged sites, 28 genes, 85 chromosomal breakpoints and 37 highly polymorphic markers . Physical mapping was both guided and confirmed using 183 bins defined by chromosomal breakpoints and by overlapping regions of YAC clones . The localization of polymorphic markers in the physical map permits the integration of physical and genetic data across the region . These data establish chromosome Xp22.3-Xp21.3 as one of the best characterized large regions in the human genome . The map should greatly facilitate finer scale mapping and sequencing as well as the identification of disease genes from this portion of the human genome.

Genome Res, 1995 Oct, 5(3), 225 - 32
Molecular cloning and RARE cleavage mapping of human 2p, 6q, 8q, 12q, and 18q telomeres; Macina RA et al.; Large terminal fragments of human chromosomes 2p, 6p, 8q, 12q, and 18q were cloned using yeast artificial chromosomes (YACs) . RecA-assisted restriction endonuclease (RARE) cleavage analysis of genomic DNA samples from II unrelated individuals using YAC-derived probes confirmed the telomeric localizations of the half-YACs studied . The cloned fragments provide telomeric closure of maps for the respective chromosome arms and will supply the reagents needed for analyzing and sequencing these distal subtelomeric regions.

Biol Chem Hoppe Seyler, 1995 Oct, 376(10), 617 - 25
Filament-specific expression of a cellulase gene in the dimorphic fungus Ustilago maydis; Schauwecker F et al.; The phytopathogenic fungus Ustilago maydis exists in a yeast-like haploid form and as a filamentous dikaryon . Only the dikaryon can infect corn plants . We have isolated a gene, egl1, that is not expressed in haploid cells but strongly induced in the filament . Molecular and biochemical analyses revealed that egl1 encodes a cellulase . By immunogold labelling, secreted protein could be detected at the hyphal tip . Mutants deleted for egl1 are viable and are affected neither in filament formation nor in pathogenic development under the conditions tested.

J Biomol Struct Dyn, 1995 Oct, 13(2), 285 - 99
A thermodynamic and mutational analysis of an RNA purine loop as a protein binding site; White SA et al.; The thermal stability and protein binding of a 36 nucleotide RNA hairpin containing an internal loop were studied under various solution conditions . Yeast ribosomal protein L32 binds to its transcript and small RNAs which reproduce the L32 transcript's secondary structure have been examined . Replacement of the internal loop with canonical base pairs did not affect the salt dependence of the melting temperature suggesting that both molecules adopt a linear shape . Several electrostatic contacts are formed on binding to a ribosomal fusion protein, but Mg+2 is not required for binding . The RNA protein complex is stable up to 50 degrees C . Two internal loop deletion mutants have similar thermodynamic stabilities and chemical and enzymatic reactivities, but fail to bind the fusion protein . However, several of the internal loop bases of the deletion mutants are moderately reactive to chemical agents whereas the wild type loop sequence displayed a mixed pattern of protection and hyperreactivity.

J Mol Cell Cardiol, 1995 Oct, 27(10), 2409 - 13
Isolation and characterization of a 1 Mb region of 5q23.3-q31.2 surrounding the human lysyl oxidase gene; McAlinden TP et al.; Lysyl oxidase (EC 1.4.3.13) plays a pivotal role in the maintenance of tissue integrity in both the normal and pathological states . It is a member of a newly discovered gene family that exhibits a complex mode of regulation . To date the resources necessary to begin to address its regulation have not been assembled . In part, this reflects the instability of this region of the genome when cloned into cosmid vectors . The paucity of long range restriction endonuclease sites suitable for mapping this region of the genome has further hampered progress . To begin to address this issue 2 YAC clones of 920 kb and 245 kb that contain the human lysyl oxidase gene were isolated . Long range physical mapping revealed that the 245 kb clone was centrally located within the 920 kb clone . The corresponding map of this region is congruent with that observed in the human genome . Thus, these YACs faithfully represent this region of the human genome . The results of our cloning and mapping studies described in this communication should accelerate the advance of our understanding of this new connective tissue gene family.

J Biochem (Tokyo), 1995 Oct, 118(4), 855 - 61
Substrate specificity of rabbit liver metalloendopeptidase and its new fluorogenic peptide substrates; Kojima N et al.; A metalloendopeptidase (MEP) isolated from rabbit liver microsomes with substrate specificity for peptides containing Arg at the P1 and P4 positions has recently proved to be identical to soluble angiotensin-binding protein present in the cytosol . Here we describe the peptide-degrading specificity of MEP, determined using various bioactive peptides and novel fluorogenic substrates for the enzyme . MEP degraded oligopeptides, including bradykinin, alpha-neoendorphin, bovine adrenal medulla dodecapeptide, substance P, bombesin, neurotensin, and alpha-endorphin, but not polypeptides such as reduced lysozyme and histone H4, hence, MEP probably belongs to the family of endo-oligopeptidases . It cleaved most preferentially at the -Phe-Ser- bond of bradykinin (kcat/Km = 2.8 x 10(4) M-1.S-1) but did not cleave high molecular weight and low molecular weight kininogens, the precursors of bradykinin . MEP did not cleave angiotensin I, dynorphin A 1-13, somatostatin, and luteinizing hormone-releasing hormone, some of which are good substrates for metalloendopeptidase-24.15, metalloendopeptidase-24.16, N-arginine dibasic convertase, and yeast endopeptidase-24.15 related peptidase . An active site-directed inhibitor of metalloendopeptidase-24.15, N-{1-(R,S)-carboxyl-3-phenylpropyl}-Ala-Ala-Phe-p-aminobenzoate also had no effects on the amidolytic activity of MEP . Based on the cleavage sites of bioactive peptides and processing sites of vitamin K-dependent proproteins, intramolecularly quenched fluorogenic peptide substrates were newly synthesized . Among the thirteen substrates used, the most reactive was 2-aminobenzoyl-Ala-Arg-Val-Arg-Arg-Ala- Asn-Ser-2,4-dinitroanilinoethylamide (kcat/Km = 9.3 x 10(5) M-1.S-1) . An angiotensin antagonist, {Sar1, Ala8}-angiotensin II, inhibited hydrolysis of the substrate by MEP in a competitive manner (Kl = 7.6 microM) . MEP cleaved oligopeptides even on the carboxyl side of proline residue and these peptides are resistant to hydrolysis by the cytosol-derived proteasome, therefore MEP may participate in the catabolism of oligopeptides in the cytosol, together with other endo-oligopeptidases.

Curr Genet, 1995 Oct, 28(5), 484 - 90
Isolation, characterization and chromosomal mapping of an actin gene from the primitive red alga Cyanidioschyzon merolae; Takahashi H et al.; Based on the results of cytological studies, it has been assumed that Cyanidioschyzon merolae does not contain actin genes . However, Southern hybridization of C . merolae cell-nuclear DNA with a yeast actin-gene probe has been suggested the presence of an actin gene in the C . merolae genome . In the present study, an actin gene was isolated from a C . merolae genomic library using a yeast actin-gene probe . The C . merolae actin gene has no intron . The predicted actin is composed of 377 amino acids and has an estimated molecular mass of 42 003 Da . Southern hybridization indicated that the C . merolae genome contains only one actin gene . This gene is transcribed at a size of 2.4 kb . When Southern hybridization was performed with C . merolae chromosomes separated by pulsed-field gel electrophoresis, a band appeared on unseparated chromosomes XI and XII . A phylogenetic tree based on known eucaryote actin-gene sequences revealed that C . merolae diverged after the division of Protozoa, but before the division of Fungi, Animalia and Chlorophyta.

Curr Genet, 1995 Oct, 28(5), 478 - 83
Primary structure and expression pattern of the 33-kDa chitinase gene from the mycoparasitic fungus Trichoderma harzianum; Limon MC et al.; A gene (chit33) from the mycoparasitic fungus Trichoderma harzianum, coding for a chitinase of 33 kDa, has been isolated and characterized . Partial amino-acid sequences from the purified 33-kDa chitinase were obtained . The amino-terminal peptide sequence was employed to design an oligonucleotide probe and was used as a primer to isolate a 1.2-kb cDNA . The cDNA codes for a protein of 321 amino acids, which includes a putative signal peptide of 19 amino acids . All microsequenced peptides found in this sequence, indicate that this cDNA codes for the 33-kDa chitinase . A high homology (approximately 43% identity) was found with fungal and plant chitinases, including yeast chitinases . However enzyme characteristics suggest a nutritional (saprophytic or mycoparasitic), rather than a morphogenetic, role for this chitinase . The chit33 gene appears as a single copy in the T . harzianum genome, is strongly suppressed by glucose, and de-repressed under starvation conditions as well as in the presence of autoclaved mycelia and/or fungal cell walls . The 33-kDa chitinase seems to be very stable except under starvation conditions . The independent regulation of each of the chitinases in T . harzianum indicates different specific roles.

Mol Cell Probes, 1995 Oct, 9(5), 361 - 70
Molecular characterization of further dystrophin gene microsatellites; King SC et al.; Microsatellites of the dystrophin gene have been used extensively in the genetic analysis of Duchenne and Becker muscular dystrophy families . The microsatellites that have been reported to date are clustered within disparate regions of the dystrophin gene, specifically at the 5'-end and in the central rod-domain . YACs encompassing the gene were screened for further microsatellites to improve the density of available genetic markers . Four microsatellites were localized to defined regions of the dystrophin gene by the analysis of patient DNA samples, somatic cell hybrids and YACs . In addition, varying combinations of microsatellite loci were amplified in multiplex PCRs, which complement those loci that have been studied to date.

Inflamm Res, 1995 Oct, 44(10), 423 - 33
Anti-inflammatory, analgesic, antipyretic and related properties of meloxicam, a new non-steroidal anti-inflammatory agent with favourable gastrointestinal tolerance; Engelhardt G et al.; The anti-inflammatory, analgesic and antipyretic properties of the new non-steroidal anti-inflammatory agent, meloxicam, were investigated in a variety of animal models and compared with the properties of piroxicam, diclofenac, indomethacin and several other NSAIDs . With respect to the total effect of a single oral dose, the anti-exudative effect of meloxicam on carrageenan-induced oedema in the rat exceeded that of all the NSAIDs included in the comparison . Additionally, meloxicam showed the greatest potency of all the compounds examined with respect to adjuvant-induced arthritis in the rat, the granuloma pouch model and the cotton pellet test in the rat . Unlike indomethacin, in the carrageenan pleurisy model in the rat, meloxicam caused both a dose-dependent reduction in exudate volume and also inhibition of leucocyte migration . Meloxicam showed a strong and lasting effect on inflammatory pain in the rat . Like other NSAIDs, but unlike dipyrone, meloxicam had no effect in the hot plate and tail clamp tests, which are used to identify weak central analgesic effects . Unlike dipyrone and like indomethacin, meloxicam had no effect in a model of visceral distention pain . In common with other NSAIDs, meloxicam had no influence on the body temperature of normothermic rats in the anti-inflammatory dose range, but did reduce yeast-induced fever in the rat in a dose-dependent manner . Like piroxicam, meloxicam had a uricosuric effect on rats treated with oxonic acid . Low-dose meloxicam inhibited both bradykinin-induced and PAF-induced bronchospasm in the guinea-pig, but had no effect on acetylcholine-induced bronchospasm . Piroxicam had greater ulcerogenic effects in the rat stomach than meloxicam . The therapeutic range of meloxicam in the rat, with regard to inhibition of adjuvant arthritis, was several times greater than that of piroxicam, indomethacin, diclofenac and naproxen.

Mamm Genome, 1995 Oct, 6(10), 725 - 31
A human ubiquitin conjugating enzyme, L-UBC, maps in the Alzheimer's disease locus on chromosome 14q24.3; Robinson PA et al.; We have identified a novel ubiquitin conjugating enzyme gene, L-UBC, which maps to human Chromosome (Chr) 14q24.3 . This is also the location of the major early onset familial Alzheimer's disease gene (FAD3) . L-UBC encodes a protein that demonstrates homology to the yeast ubiquitin conjugating enzyme, UBC-4, and human UbcH5 . Their functions are to ubiquitinate specific proteins targeted for degradation . The protein also exhibits very strong homology to a rabbit protein, E2-F1, which mediates p53 degradation driven by papilloma virus E6 protein in vitro . The accumulation of specific proteins that have undergone aberrant processing in neurofibrillary tangles and amyloid plaques is the classic pathological feature in brains of Alzheimer's disease patients . Abnormal ubiquitination has previously been suggested to play a role in the etiology of Alzheimer's disease . This gene therefore represents a plausible candidate gene for FAD3.

Mech Dev, 1995 Oct, 53(2), 247 - 60
Dmcdc2 kinase is required for both meiotic divisions during Drosophila spermatogenesis and is activated by the Twine/cdc25 phosphatase; Sigrist S et al.; We have analyzed the requirement for Drosophila cdc2 kinase during spermatogenesis after generating temperature-sensitive mutant lines (Dmcdc2ts) by re-constructing mutations known to result in temperature sensitivity in fission yeast cdc2+ . While meiotic spindles and metaphase plates were never formed in Dmcdc2ts mutants at high temperature, chromosomes still condensed in late spermatocytes and spermatid differentiation (sperm head and tail formation) continued . The same phenotype was also observed in twine and twine, Dmcdc2ts double mutant testes, consistent with the idea that the cdc2 kinase activity required for meiotic divisions is activated by the Twine/cdc25 phosphatase . Confirming this notion, we find that ectopic expression of the String/cdc25 phosphatase, which is known to activate the cdc2 kinase before mitosis, results in a partial rescue of meiotic divisions in twine mutant testis.

Protein Sci, 1995 Oct, 4(10), 2087 - 99
Atomic solvation parameters in the analysis of protein-protein docking results; Cummings MD et al.; Several sets of amino acid surface areas and transfer free energies were used to derive a total of nine sets of atomic solvation parameters (ASPs) . We tested the accuracy of each of these sets of parameters in predicting the experimentally determined transfer free energies of the amino acid derivatives from which the parameters were derived . In all cases, the calculated and experimental values correlated well . We then chose three parameter sets and examined the effect of adding an energetic correction for desolvation based on these three parameter sets to the simple potential function used in our multiple start Monte Carlo docking method . A variety of protein-protein interactions and docking results were examined . In the docking simulations studied, the desolvation correction was only applied during the final energy calculation of each simulation . For most of the docking results we analyzed, the use of an octanol-water-based ASP set marginally improved the energetic ranking of the low-energy dockings, whereas the other ASP sets we tested disturbed the ranking of the low-energy dockings in many of the same systems . We also examined the correlation between the experimental free energies of association and our calculated interaction energies for a series of proteinase-inhibitor complexes . Again, the octanol-water-based ASP set was compatible with our standard potential function, whereas ASP sets derived from other solvent systems were not.

Protein Sci, 1995 Oct, 4(10), 1985 - 97
Water molecules participate in proteinase-inhibitor interactions: crystal structures of Leu18, Ala18, and Gly18 variants of turkey ovomucoid inhibitor third domain complexed with Streptomyces griseus proteinase B; Huang K et al.; Crystal structures of the complexes of Streptomyces griseus proteinase B (SGPB) with three P1 variants of turkey ovomucoid inhibitor third domain (OMTKY3), Leu18, Ala18, and Gly18, have been determined and refined to high resolution . Comparisons among these structures and of each with native, uncomplexed SGPB reveal that each complex features a unique solvent structure in the S1 binding pocket . The number and relative positions of water molecules bound in the S1 binding pocket vary according to the size of the side chain of the P1 residue . Water molecules in the S1 binding pocket of SGPB are redistributed in response to the complex formation, probably to optimize hydrogen bonds between the enzyme and the inhibitor . There are extensive water-mediated hydrogen bonds in the interfaces of the complexes . In all complexes, Asn 36 of OMTKY3 participates in forming hydrogen bonds, via water molecules, with residues lining the S1 binding pocket of SGPB . For a homologous series of aliphatic straight side chains, Gly18, Ala18, Abu18, Ape18, and Ahp18 variants, the binding free energy is a linear function of the hydrophobic surface area buried in the interface of the corresponding complexes . The resulting constant of proportionality is 34.1 cal mol-1 A-2 . These structures confirm that the binding of OMTKY3 to the preformed S1 pocket in SGPB involves no substantial structural disturbances that commonly occur in the site-directed mutagenesis studies of interior residues in other proteins, thus providing one of the most reliable assessments of the contribution of the hydrophobic effect to protein-complex stability.

Biosci Biotechnol Biochem, 1995 Oct, 59(10), 1913 - 20
Histological study of iron deposits in selenium-deficient rats; Chareonpong-Kawamoto N et al.; Previous studies have shown that selenium (Se) deficiency is associated with hematological abnormalities, which may result in an increased distribution of iron in various tissues . This report describes histological studies of the location of excess iron deposits in tissues . Male Wistar rats were fed a Torula yeast-based Se-deficient {Se(-)} or Se-adequate {Se(+); containing 0.1 mg Se/kg as sodium selenite} diet for 8 or 82 weeks . Excised tissues were embedded in either paraffin or epoxy resin . A dramatic increase was observed in iron deposition in the liver and kidneys of rats on the Se(-) diet . Prussian blue-stained sections under the light microscope showed iron deposits in the parenchymal cells and Kupffer cells of liver and in the proximal tubules of kidneys . The liver and kidneys of Se(-) rats had considerably altered morphology: lysosomes were enlarged and contained electron-dense areas . X-Ray microanalysis showed that the areas that corresponded to the lysosomes contained iron . No iron deposits were observed in sections of kidney and liver from rats fed the Se(+) diet . Thus, these studies identified subcellular sites of iron deposition in the liver and kidneys of Se(-) rats . These iron deposits may be an important factor in the pathogenesis of Se deficiency.

Z Lebensm Unters Forsch, 1995 Oct, 201(4), 399 - 401
Use of UV photography to identify aflatoxin-producing strains of Aspergillus flavus and A . parasiticus; Cvetnic Z et al.; UV photography in glucose, yeast extract (GY) agar medium was tested as a simple and rapid method for the distinction of aflatoxin-positive from aflatoxin-negative strains of Aspergillus flavus and A . parasiticus . In the UV photographs aflatoxin-producing moulds were identified as grey or black colonies, whereas aflatoxin-nonproducing moulds appeared as white colonies . Of the aflatoxin-positive strains detected by the UV photographic method, 10% was confirmed by extraction of the GY agar medium and mould mycelium in chloroform, extracts which were analysed subsequently using thin-layer chromatography . Confirmation of aflatoxigenic strains was achieved by biosynthesis on liquid medium yeast extract sucrose (YES) broth.

Eur J Biochem, 1995 Oct 1, 233(1), 277 - 82
Valyl-tRNA synthetase from Artemia . Purification and association with elongation factor 1; Brandsma M et al.; Two components of the protein biosynthetic machinery, valyl-transfer RNA synthetase (VRS) and elongation factor 1 (EF-1), have been isolated as a complex from several mammalian tissues . However, yeast VRS, which lacks an amino-terminal extension, does not associated with EF-1 . We purified VRS from the brine shrimp Artemia and investigated its interaction with EF-1 . Western blotting of crude Artemia extracts revealed the presence of two forms of VRS, differing in size and capacity to associate with EF-1 . About 80% of the total VRS corresponds to a polypeptide of 130 kDa which behaves as a monomer upon gel filtration . Only the larger form of 140 kDa coelutes, cosediments and co-immunoprecipitates with the EF-1 alpha 2 beta gamma delta complex . The ratio of the two forms of VRS remains constant throughout early development . The possible origin and mode of expression of the two forms of VRS present in Artemia are discussed.

Plant Cell, 1995 Oct, 7(10), 1611 - 23
Composite structure of auxin response elements; Ulmasov T et al.; The auxin-responsive soybean GH3 gene promoter is composed of multiple auxin response elements (AuxREs), and each AuxRE contributes incrementally to the strong auxin inducibility to the promoter . Two independent AuxREs of 25 bp (D1) and 32 bp (D4) contain the sequence TGTCTC . Results presented here show that the TGTCTC element in D1 and D4 is required but not sufficient for auxin inducibility in carrot protoplast transient expression assays . Additional nucleotides upstream of TGTCTC are also required for auxin inducibility . These upstream sequences showed constitutive activity and no auxin inducibility when part or all of the TGTCTC element was mutated or deleted . In D1, the constitutive element overlaps the 5' portion of TGTCTC; in D4, the constitutive element is separated from TGTCTC . An 11-bp element in D1, CCTCGTGTCTC, conferred auxin inducibility to a minimal cauliflower mosaic virus 35S promoter in transgenic tobacco seedlings as well as in carrot protoplasts (i.e., transient expression assays) . Both constitutive elements bound specifically to plant nuclear proteins, and the constitutive element in D1 bound to a recombinant soybean basic leucine zipper transcription factor with G-box specificity . To demonstrate further the composite nature of AuxREs and the ability of the TGTCTC element to confer auxin inducibility, we created a novel AuxRE by placing a yeast GAL4 DNA binding site adjacent to the TGTCTC element . Expression of a GAL4-c-Rel transactivator in the presence of this novel AuxRE resulted in auxin-inducible expression . Our results indicate that at least some AuxREs have a composite structure consisting of a constitutive element adjacent to a conserved TGTCTC element that confers auxin inducibility.

Plant Mol Biol, 1995 Oct, 29(2), 293 - 301
Immunological evidence for accumulation of two high-molecular-weight (104 and 90 kDa) HSPs in response to different stresses in rice and in response to high temperature stress in diverse plant genera; Pareek A et al.; Rice seedlings accumulate stainable amounts of the 104 and 90 kDa polypeptides in response to high temperature stress . We have purified and raised highly specific polyclonal antisera against both of these polypeptides . In western blotting experiments, we find that these proteins are accumulated to different extents in rice seedlings subjected to salinity (NaCl), water stress, low-temperature stress and exogenous abscisic acid application . These proteins also accumulated when rice seedlings grown in pots under natural conditions were subjected to water stress by withholding watering . Seedlings of Triticum aestivum, Sorghum bicolor, Pisum sativum, Zea mays, Brassica juncea and mycelium of Neurospora crassa showed accumulation of the immunological homologues of both the 104 and the 90 kDa polypeptides, in response to high-temperature stress . We have earlier shown that shoots of rice seedlings exposed to heat shock accumulate a 110 kDa polypeptide which is an immunological homologue of the yeast HSP 104 (Singla and Grover, Plant Mol Biol 22: 1177-1180, 1993) . Employing anti-rice HSP 104 antibodies and anti-yeast HSP 104 antibodies together, we provide evidence that rice HSP 104 is different from the earlier characterized rice HSP 110.

Plant Mol Biol, 1995 Oct, 29(1), 37 - 51
Isolation and characterization of six heat shock transcription factor cDNA clones from soybean; Czarnecka-Verner E et al.; Thermal stress in soybean seedlings causes the activation of pre-existing heat shock transcription factor proteins (HSFs) . Activation results in the induction of DNA binding activity which leads to the transcription of heat shock genes . From a soybean cDNA library we have isolated cDNA clones corresponding to six HSF genes . Two HSF genes are expressed constitutively at the transcriptional level, and the remaining four are heat-inducible . Two of the heat inducible genes are also responsive to cadmium stress . Comparative analysis of HSF sequences indicated higher conservation of the DNA binding domain among plant HSFs than those from yeast or other higher eukaryotes . The putative plant HSF oligomerization domain contains hydrophobic heptapeptide repeats characteristic of coiled coils and seems to exist in two structural variants . The carboxy-terminal domains are reduced in size and the C-terminal heptad repeat is degenerate.

Br J Dermatol, 1995 Oct, 133(4), 537 - 41
Adherence of Malassezia isolates to human keratinocytes in vitro--a study of HIV-positive patients with seborrhoeic dermatitis; Schechtman RC et al.; Adherence of Malassezia yeast cells to human keratinocytes was assessed by a novel technique using double-sided Sellotape . Although adherence using double-sided Sellotape is still merely a model for in vivo adherence, it approximates to the conditions found on the skin surface . There were no differences in adhesive properties to human keratinocytes between Malassezia strains originating from HIV-positive and HIV-negative patients with seborrhoeic dermatitis, nor was there a relationship between the severity of seborrhoeic dermatitis and in vitro adherence to human keratinocytes.

Arch Biochem Biophys, 1995 Oct 1, 322(2), 361 - 8
Terminal marking of triosephosphate isomerase: consequences of deamidation; Sun AQ et al.; Mammalian triosephosphate isomerase spontaneously deamidates at Asn71 and Asn15 located at the subunit interface of the isologous dimer . These deamidations have been proposed to constitute the terminal marking event in the degradation of the enzyme . A series of physical and chemical studies detailed here reveals that the overall structure of the enzyme is substantially altered by these deamidations . The far-uv CD spectra show a 30% lower secondary structure with a blue shifted ellipticity minimum and increased fluorescence (10-22%) with a red-shifted emission maximum (8.7-15.6 nm) indicates exposure of tryptophans to a more polar environment . Increased binding of the fluorescent hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid to the deamidated enzyme corroborates these spectral observations and also suggests that the hydrophobic residues at the subunit interface are exposed as a result of the deamidation . Decreased subunit cross-linking (80 vs 20%) of the deamidated enzyme by the bifunctional reagent ethylene glycolbis (succinimidylsuccinate) also indicates a loosening of the two subunits at the interface . These structural changes are accompanied by a decreased thermal stability (3.1 degrees C lower Tm) and an increased susceptibility to dissociation in urea . The terminal marking also results in the generation of new proteolytic sites and increases the susceptibility to proteolysis . Hybrid dimers from rabbit and yeast (lacking Asn71) showed that deamidation of the rabbit Asn71-yeast Asn15 pair does not accelerate deamidation of the remaining rabbit Asn15 site, indicating that deamidation of Asn71 is a prerequisite for deamidation of Asn15 . These studies are consistent with the proposal that the specific deamidations at the subunit interface cause significant structural changes which lead to degradation of the protein.

Virology, 1995 Oct 1, 212(2), 422 - 8
The influenza virus NS1 protein forms multimers in vitro and in vivo; Nemeroff ME et al.; The NS1 protein of the influenza A virus inhibits both the nuclear export of mRNA and pre-mRNA splicing . Two functional domains, an RNA-binding domain and an effector domain, have been identified in this protein . Here we demonstrate that the NS1 protein exists as a dimer in vitro both in the absence of its RNA target and when it is bound to a specific RNA target, U6 snRNA . This indicates that it is most likely the dimer that binds to the RNA target . Mutational analysis indicated that the RNA-binding and dimerization domains are coincident . Multimerization also occurs in vivo, as assayed using the yeast two-hybrid system . In contrast to the situation in vitro, multimerization in vivo was mediated by not only the RNA-binding domain but also the effector domain . This suggests that multimerization in vivo involves a cellular protein cofactor that bridges more than one NS1 protein molecule together via their effector domains.

Mol Cell Biol, 1995 Oct, 15(10), 5777 - 88
mRNA decay mediated by two distinct AU-rich elements from c-fos and granulocyte-macrophage colony-stimulating factor transcripts: different deadenylation kinetics and uncoupling from translation; Chen CY et al.; Poly(A) tail removal is a critical first step in the decay pathway for many yeast and mammalian mRNAs . Poly(A) shortening rates can be regulated by cis-acting sequences within the transcribed portion of mRNA, which in turn control mRNA turnover rates . The AU-rich element (ARE), found in the 3' untranslated regions of many highly labile mammalian mRNAs, is a well-established example of this type of control . It represents the most widespread RNA stability determinant among those characterized in mammalian cells . Here, we report that two structurally different AREs, the c-fos ARE and the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE, both direct rapid deadenylation as the first step in mRNA degradation, but by different kinetics . For c-fos-ARE-mediated decay, the mRNA population undergoes synchronous poly(A) shortening and is deadenylated at the same rate, implying the action of distributive or nonprocessive ribonucleolytic digestion of poly(A) tails . In contrast, the population of granulocyte-macrophage colony-stimulating factor ARE-containing mRNAs is deadenylated asynchronously, with the formation of fully deadenylated intermediates, consistent with the action of processive ribonucleolytic digestion of poly(A) tails . An important general implication of this finding is that different RNA-destabilizing elements direct deadenylation either by modulating the processivity at which a single RNase functions or by recruiting kinetically distinct RNases . We have also employed targeted inhibition of translation initiation to demonstrate that the RNA-destabilizing function of both AREs can be uncoupled from translation by ribosomes . In addition, a blockade of ongoing transcription has been used to further probe the functional similarities and distinctions of these two AREs . Our data suggest that the two AREs are targets of two distinct mRNA decay pathways . A general model for ARE-mediated mRNA degradation involving a potential role for certain heterogeneous nuclear ribonucleoproteins and ARE-binding proteins is proposed.

Mol Cell Biol, 1995 Oct, 15(10), 5214 - 25
A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function; Catling AD et al.; Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4 . Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function . Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation . In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts . Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts . Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation . Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts . These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

J Leukoc Biol, 1995 Oct, 58(4), 382 - 90
Natural resistance to infection with intracellular parasites: molecular genetics identifies Nramp1 as the Bcg/Ity/Lsh locus; Vidal S et al.; Natural resistance to infection with intracellular parasites is controlled in the mouse by the expression of a locus or group of loci on chromosome 1 alternatively named Bcg, Lsh, and Ity . Bcg affects the capacity of mature tissue macrophages to restrict the intracellular proliferation of ingested parasites in the reticuloendothelial organs of the host during the early phase of infection . This review summarizes our molecular genetic approach to the isolation and characterization of the Bcg locus . We have used a positional cloning strategy based on genetic and physical mapping, YAC cloning, and exon trapping to isolate a candidate gene for Bcg, named Nramp1, which codes for a macrophage-specific polytopic protein with 12 predicted transmembrane domains and a consensus transport motif . Sequence analysis of Nramp1 cDNA clones from 27 Bcgs and Bcgr mouse strains reveals that susceptibility to infection (Bcgs) is associated with a single nonconservative Gly to Asp substitution at position 169 within predicted transmembrane domain 4 of the Nramp protein . Cloning experiments and homology search in available databases demonstrated that the Nramp1 gene belongs to a small gene family with several members in vertebrates and in such distantly related species as yeast and plants . Nramp proteins share a remarkable degree of similarity, with strong amino acid sequence conservation in the transmembrane domains, suggesting a common transport function for the Nramp family . Finally, we generated Nramp1-/- gene knockout mice, and analysis of their phenotypic characteristics established that (1) Nramp1 plays a key role in natural defense against infection with intracellular parasites and therefore demonstrated allelism between Nramp1 and Bcg/Ity/Lsh, (2) Nramp1 functions by a novel cytocidal/cytostatic mechanism distinct from those expressed by the activated macrophage, and (3) the Nramp1Asp169 allele of Bcgs inbred strains is a null allele, pointing to a critical role of this residue in Nramp1 function.

Hum Genet, 1995 Oct, 96(4), 477 - 80
Subregional mapping of the human gonadotropin-releasing hormone receptor (GnRH-R) gene to 4q between the markers D4S392 and D4S409; Kottler ML et al.; We have isolated nine yeast artificial chromosomes (YACs) containing the gene that encodes the human gonadotropin-releasing hormone receptor (GnRH-R) gene by screening the YAC library of the Centre d'Etude du Polymorphisme Humain (Hopital Saint-Louis, Paris, France) by the use of the polymerase chain reaction . We defined the location of the GnRH-R gene relative to 4q microsatellite markers D4S392 and D4S409 . The genetic positions of these markers on chromosome 4 are 76 and 77 cM, respectively . This location was further established by chromosomal in situ hybridization.

FASEB J, 1995 Oct, 9(13), 1311 - 8
Ras target proteins in eukaryotic cells; Marshall MS; The 21 kDa Ras proteins are well known for their regulatory role in oncogenic, mitogenic, and developmental signaling pathways . Less well understood are the downstream signal transduction cascades initiated by Ras in response to external stimuli . Only recently have many diverse studies in lower eukaryotes and vertebrates converged to demonstrate that Ras directly regulates multiple signaling pathways . In most eukaryotes, Ras functions as a positive regulator of an ERK/MAPK signal transduction cascade through the activation of a MEKK . In mammalian cells the primary Ras-responsive MEKK is the protein kinase Raf . Although Raf remains the most significant mediator of Ras signaling in most model systems, it does not explain all the biochemical responses observed in cells with activated Ras proteins . Yeast two hybrid and GST-fusion protein binding studies have identified new proteins distinct from Raf that could interact with Ras in other signaling pathways . In addition to Raf, other potential Ras target proteins include MEKK1, PI(3)K, p120GAP, ralGDS, and PKC zeta . This review will attempt to summarize the current literature of accepted and potential Ras-dependent signaling proteins in both lower eukaryotes and vertebrates.

Clin Exp Immunol, 1995 Oct, 102(1), 65 - 70
Antifungal mechanisms of activated murine bronchoalveolar or peritoneal macrophages for Histoplasma capsulatum; Brummer E et al.; The first line of defence against natural infection by Histoplasma capsulatum (Hc) consists of bronchoalveolar macrophages (BAM) and an early inflammatory response in the lungs . Little is known about the interaction of BAM and Hc, consequently we studied murine BAM in vitro to assess their role in the pulmonary defence in histoplasmosis . A short-term 3-h assay was used to measure fungicidal activity of control BAM and interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-activated BAM . Fungistatic activity of BAM was determined with a 24-h assay . A method devised for measuring colony-forming units (CFU) of non-ingested non-adherent and adherent ingested yeast cells of Hc in BAM cocultures was used . Activated BAM killed Hc (reduced inoculum CFU by 25 +/- 12%; n = 4) . The fungicidal activity of BAM was abrogated by 0.2 mM NG-monomethyl-L-arginine (NMMA) or catalase but not by superoxide dismutase . In fungistatic assays activated BAM inhibited multiplication of Hc by 61 +/- 4% (n = 3) compared with cocultures with control BAM . However, Hc multiplied 100% more in control BAM cocultures than in medium alone . Data indicated that this was due to advantages that Hc has in the intracellular environment . Only NMMA inhibited fungistatic activity of activated BAM . In experiments with peritoneal macrophages (PM), results similar to those with BAM were obtained . In conclusion, activated BAM and PM kill yeast cells of Hc by a mechanism dependent on hydrogen peroxide and products of the nitric oxide synthase (NOS) pathway, whereas fungistasis depends only on products of the NOS pathway.

Science, 1995 Sep 29, 269(5232), 1869 - 72
A specialized pathway affecting virulence glycoconjugates of Leishmania; Descoteaux A et al.; For virulence and transmission, the protozoan parasite Leishmania must assemble a complex glycolipid on the cell surface, the lipophosphoglycan (LPG) . Functional complementation identified the gene LPG2, which encodes an integral Golgi membrane protein implicated in intracellular compartmentalization of LPG biosynthesis . Ipg2- mutants lack only characteristic disaccharide-phosphate repeats, normally present on both LPG and other surface or secreted molecules considered critical for infectivity . In contrast, a related yeast gene, VAN2/VRG4, is essential and required for general Golgi function . These results suggest that LPG2 participates in a specialized virulence pathway, which may offer an attractive target for chemotherapy.

Ann N Y Acad Sci, 1995 Sep 29, 764, 62 - 71
Characterization and genomic mapping of a novel leader peptide associated with the human VH4-21 (VH4-34) gene segment; Widhopf GF 2nd et al.; The human IgVH locus is located on chromosome 14, band q32 and spans approximately 1 mb . Within this locus are approximately 120 VH gene segments that are subdivided into six to seven families based on sequence homology of their coding regions . VH4-21 (VH4-34) is a member of the VH4 family, a family that contains 10 to 15 members . It is expressed in a variety of circumstances including early fetal development, the autoantibody repertoire, and in a highly restricted manner in antibodies that recognize alloantigens on the surface of human red blood cells . Most interesting, however, is the expression of this gene segment in T cells as a semi-germline transcript in conjunction with a nontraditional VH leader peptide . This nonhydrophobic leader sequence, termed "Et" for exon in T cells, has previously been shown to reside within the VH locus . Using YAC and P1 clones, we have identified two copies of this exon, both of which are located in the region of the locus that contains VH4-24 (VH4-34) . Characterization of the two exons suggests that they arose by duplication, as flanking DNA is almost identical over a distance of > 5 kb . Preliminary data suggests they are both located > 20 kb upstream of VH4-21 (VH4-34).

Ann N Y Acad Sci, 1995 Sep 29, 764, 525 - 35
Production of antigen-specific human antibodies from mice engineered with human heavy and light chain YACs; Jakobovits A et al.; Our paper describes the introduction of large fragments of both the human heavy and light chain Ig genes into the mouse germline to create a mouse strain capable of producing a broad repertoire of antigen-specific, fully human antibodies . The human immunoglobulin gene sequences were functional in the context of the mouse machinery for antibody recombination and expression, either in the presence or absence of functional endogenous genes . This was demonstrated by their ability to undergo diverse rearrangement, to be expressed at significant levels, and to exclude expression of mouse immunoglobulins irrespective of their copy number or site of integration . The decrease in susceptibility to influence by adjacent genomic sequences may reflect the greater size, variable gene content, or structural integrity of the human Ig YACs and/or the presence of unidentified but important regulatory elements needed for optimal expression of the human immunoglobulin genes and their correct regulation . Our results show that mouse B cells coexpressing human heavy and kappa chains, upon immunization, can produce antigen-specific, fully human antibodies . Furthermore, the human heavy and kappa chain YACs induced differentiation and maturation of the growth-arrested B-cell lineage in mice with inactivated endogenous Ig genes, leading to the production of a diverse repertoire of fully human antibodies at levels approaching those in normal serum . These results suggest the potential value of these mice as a source of fully human antibodies for human therapy . Furthermore, it is expected that such mice would lack immunological tolerance to and thus readily yield antibodies to human proteins, which may constitute an important class of targets for monoclonal antibody therapy . Our findings suggest that the introduction of even larger portions of the human heavy and light chain loci, which should be achievable with the ES cell-yeast spheroplast fusion technology described, will result in strains of mice ultimately capable of recapitulating the full antibody repertoire characteristic of the human humoral response to infection and immunization . The present and future mouse strains may prove to be valuable tools for studying the molecular mechanisms and regulatory sequences influencing the programmed assembly and expression of human antibodies in the normal immune response, as well as the abnormal response characteristic of autoimmune disease and other disorders . The strategy we have described for the introduction of large segments of the human genome into mice in conjunction with the inactivation of the corresponding mouse loci may also have broad applicability to the investigation of other complex or uncharacterized loci.

Nature, 1995 Sep 28, 377(6547 Suppl), 367 - 79
A high-density YAC contig map of human chromosome 22; Collins JE et al.; We have constructed a high-resolution clone map of human chromosome 22 which integrates the available physical and genetic information, establishing a single consensus . The map consists of all classes of DNA landmarks ordered on 705 yeast artificial chromosomes (YACs) at an average landmark density of more than one per 70 kilobases . This map represents the practical limits of currently available YAC resources and provides the basis for determination of the entire gene content and genomic DNA sequence of human chromosome 22.

Nature, 1995 Sep 28, 377(6547 Suppl), 335 - 65
An integrated physical map of human chromosome 16; Doggett NA et al.; We describe an integrated physical, genetic and cytogenetic map of human chromosome 16 comprising both a low-resolution megaYAC map and a high-resolution cosmid contig/miniYAC map, which provides nearly complete coverage of the euchromatic arms of the chromosome . The physical map is anchored to a high-resolution cytogenetic breakpoint map and is integrated with genetic and gene transcript maps of the chromosome by sequence-tagged sites and clone hybridizations.

Nature, 1995 Sep 28, 377(6547 Suppl), 321 - 33
A second-generation YAC contig map of human chromosome 12; Krauter K et al.; Human chromosome 12 constitutes approximately 4.5% of the human genome and has an estimated size of 135 million base pairs (Mb) . We have started to construct a high-resolution physical map of chromosome 12 as overlapping yeast artificial chromosomes (YACs), using as a foundation the first-generation physical map of this chromosome covers nearly 102 Mb of DNA and includes 426 highly polymorphic, monomorphic and gene-based markers . We also mapped 119 of the YACs, most of which are part of the physical map, by cytogenetic methods . Thus the map integrates genetic, physical and cytogenetic data and provides information about the organization of this chromosome and will help in the localization and cloning of disease-related genes . The strategy used here to generate the chromosome-12 map could be applied for the rapid construction of physical and expression maps for other human chromosomes.

Nature, 1995 Sep 28, 377(6547 Suppl), 299 - 319
A second-generation YAC contig map of human chromosome 3; Gemmill RM et al.; A map of human chromosome 3 which integrates both physical and genetic data has been developed from the fusion of two large collections of markers and corresponding yeast artificial chromosome (YAC) clones . The map contains 972 megabase-sized YACs identified with 593 primary markers, of which 162 are highly polymorphic sequence-tagged sites (STSs) and form a closely spaced genetic linkage map; the remaining markers are hybridization-based . Chromosome 3 is now represented by 24 large YAC contigs whose order and orientation is largely known . The map generated by fusion of these hybridization- and STS-based datasets covers about 80% (over 160 megabases) of the chromosome and will provide the foundation necessary for rapid development of a detailed genetic understanding for this large autosome.

Nature, 1995 Sep 28, 377(6547 Suppl), 175 - 297
A YAC contig map of the human genome; Chumakov IM et al.; A yeast artificial chromosome library containing 33,000 clones with an average insert size of one megabase of human genomic DNA was extensively analysed by several different procedures for detecting overlaps and positional information . We developed an analysis strategy that resulted, after confirmatory tests, in a YAC contig map reliably covering about 75% of the human genome in 225 contigs having an average size of about ten megabases.

Proc Natl Acad Sci U S A, 1995 Sep 26, 92(20), 9373 - 7
Plant members of a family of sulfate transporters reveal functional subtypes; Smith FW et al.; Three plant sulfate transporter cDNAs have been isolated by complementation of a yeast mutant with a cDNA library derived from the tropical forage legume Stylosanthes hamata . Two of these cDNAs, shst1 and shst2, encode high-affinity H+/sulfate cotransporters that mediate the uptake of sulfate by plant roots from low concentrations of sulfate in the soil solution . The third, shst3, represents a different subtype encoding a lower affinity H+/sulfate cotransporter, which may be involved in the internal transport of sulfate between cellular or subcellular compartments within the plant . The steady-state level of mRNA corresponding to both subtypes is subject to regulation by signals that ultimately respond to the external sulfate supply . These cDNAs represent the identification of plant members of a family of related sulfate transporter proteins whose sequences exhibit significant amino acid conservation in filamentous fungi, yeast, plants, and mammals.

Biochemistry, 1995 Sep 26, 34(38), 12435 - 44
Double-stranded damage of DNA.RNA hybrids by neocarzinostatin chromophore: selective C-1' chemistry on the RNA strand; Zeng X et al.; Glutathione-activated neocarzinostatin chromophore generates bistranded lesions in the hybrid formed by yeast tRNA(phe) and DNA complementary to its 31-mer 3' terminus . To elucidate the chemistry of the RNA cleavage reaction and to show that the lesions are double-stranded (ds), a series of shorter oligoribonucleotides containing the target sequence r(AGAAUUC).(GAATTCT) (underlining indicates major attack site) was studied as substrates . In addition to cleavage at both U residues, major damage was produced in the form of an abasic site at the U residues . Evidence for abasic site formation on the RNA strand was obtained from sequencing-gel analysis and measurement of uracil base release . Initial evidence for the ds nature of the damage came from experiments in which 2'-O-methyluridine was substituted for uridine in the RNA at one or both of the target sites . The site containing the substitution was not a target for cleavage or abasic site formation, and the particular T residue, staggered two nucleotides in the 3' direction on the complementary DNA strand, was cleaved significantly less . These studies were valuable in identifying the DNA ds partner of the RNA attack site . Direct evidence for ds lesions came from analysis of the products from a hairpin oligonucleotide construct in which the RNA and DNA strands were linked by four T residues and contained an internal 32P label at the 3' end of the RNA strand . Substitution of deuterium for hydrogen at the C-1' position of the U residues led to a substantial isotope effect (k1H/k2H = 3) upon the formation of the RNA abasic lesion and the RNA cleavage products, providing conclusive evidence for selective 1' chemistry . On the other hand, cleavage at the T residues on the complementary DNA strand involved C-5' hydrogen abstraction, as was also true for the T residue in an oligodeoxynucleotide analogue of the RNA strand . Chemical mechanisms to account for the RNA cleavage and abasic site formation via C-1' hydrogen abstraction are proposed.

Nucleic Acids Res, 1995 Sep 25, 23(18), 3638 - 41
Identification of the factors that interact with NCBP, an 80 kDa nuclear cap binding protein; Kataoka N et al.; It has been shown that the monomethylated cap structure plays important roles in pre-mRNA splicing and nuclear export of RNA . As a candidate for the factor involved in these nuclear events we have previously purified an 80 kDa nuclear cap binding protein (NCBP) from a HeLa cell nuclear extract and isolated its full-length cDNA . In this report, in order to obtain a clue to the cellular functions of NCBP, we attempted to identify a factor(s) that interacts with NCBP . Using the yeast two-hybrid system we isolated three clones from a HeLa cell cDNA library . We designated the proteins encoded by these clones NIPs (NCBP interacting proteins) . NIP1 and NIP2 have an RNP consensus-type RNA binding domain, whereas NIP3 contains a unique domain of Arg-Glu or Lys-Glu dipeptide repeats . We also show that NCBP requires NIP1 for binding to the cap structure . Possible roles of NIPs in cap-dependent nuclear processes are discussed.

Science, 1995 Sep 22, 269(5231), 1737 - 40
Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95; Kornau HC et al.; The N-methyl-D-aspartate (NMDA) receptor subserves synaptic glutamate-induced transmission and plasticity in central neurons . The yeast two-hybrid system was used to show that the cytoplasmic tails of NMDA receptor subunits interact with a prominent postsynaptic density protein PSD-95 . The second PDZ domain in PSD-95 binds to the seven-amino acid, COOH-terminal domain containing the terminal tSXV motif (where S is serine, X is any amino acid, and V is valine) common to NR2 subunits and certain NR1 splice forms . Transcripts encoding PSD-95 are expressed in a pattern similar to that of NMDA receptors, and the NR2B subunit co-localizes with PSD-95 in cultured rat hippocampal neurons . The interaction of these proteins may affect the plasticity of excitatory synapses.

Cell, 1995 Sep 22, 82(6), 885 - 93
Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes; Latterich M et al.; The fusion of endoplasmic reticulum (ER) membranes in yeast is an essential process required for normal progression of the nuclear cell cycle, karyogamy, and the maintenance of an intact organellar compartment . We showed previously that this process requires a novel fusion machinery distinct from the classic membrane docking/fusion machinery containing Sec17p (alpha-SNAP) and Sec18p (NSF) . Here we show that Cdc48p, a cell-cycle protein with homology to Sec18p, is required in ER fusion . A temperature-sensitive cdc48 mutant is conditionally defective in ER fusion in vitro . Addition of purified Cdc48p restores the fusion of isolated cdc48 mutant ER membranes . We propose that Cdc48p is part of an evolutionarily conserved fusion/docking machinery involved in multiple homotypic fusion events.

Nature, 1995 Sep 21, 377(6546), 246 - 8
Distinct functions for the two importin subunits in nuclear protein import; Gorlich D et al.; The import of nuclear proteins proceeds through the nuclear pore complex and requires nuclear localization signals (NLSs), energy and soluble factors, namely importin-alpha (M(r) 60K), importin-beta (90K) and Ran . Importin-alpha is primarily responsible for NLS recognition and is a member of a protein family that includes the essential yeast nuclear pore protein SRP1p (ref . 16) . As the first event, the complex of importin-alpha and importin-beta binds the import substrate in the cytosol . Here we show that this nuclear pore targeting complex initially docks as a single entity to the nuclear pore via importin-beta . Then the energy-dependent, Ran-mediated translocation through the pore results in the accumulation of import substrate and importin-alpha in the nucleus . In contrast, importin-beta accumulates at the nuclear envelope, but not in the nucleoplasm . Immunoelectron microscopy detects importin-beta on both sides of the nuclear pore . This suggests that the nuclear pore targeting complex might move as a single entity from its initial docking site through the central part of the nuclear pore before it disassembles on the nucleoplasmic side.

Genomics, 1995 Sep 20, 29(2), 512 - 25
An STS content map of human chromosome 11: localization of 910 YAC clones and 109 islands; Quackenbush J et al.; Physical mapping of human chromosomes at a resolution of 100 kb to 1 Mb will provide important reagents for gene identification and framework templates for ultimately determining the complete DNA sequence . Sequence-tagged site (STS) content mapping, coupled with large fragment cloning in yeast artificial chromosomes, provides an efficient mechanism for producing first-generation, low-resolution maps of human chromosomes . Previously, we produced a set of standardized STSs for human chromosome 11 regionally localized by fluorescence in situ hybridization or somatic cell hybrid analysis . In this paper, we used these as well as other STS content, and identify 109 islands spanning an estimated 218 Mb on the 126-Mb chromosome . Since about 62% of the islands contain markers ordered on chromosome 11 by genetic or radiation hybrid analysis, this data set represents a first-order approximation of a physical map of human chromosome 11 . This set of clones, contigs, and associated STSs will provide the material for the production of a continuous overlapping set of YACs as well for high-resolution physical mapping based upon sampled and complete DNA sequencing.

Genomics, 1995 Sep 20, 29(2), 496 - 502
Construction of two YAC contigs in human Xp11.23-p11.22, one encompassing the loci OATL1, GATA, TFE3, and SYP, the other linking DXS255 to DXS146; Fisher SE et al.; We have constructed two YAC contigs in the Xp11.23-p11.22 interval of the human X chromosome, a region that was previously poorly characterized . One contig, of at least 1.4 Mb, links the pseudogene OATL1 to the genes GATA1, TFE3, and SYP and also contains loci implicated in Wiskott-Aldrich syndrome and synovial sarcoma . A second contig, mapping proximal to the first, is estimated to be over 2.1 Mb and links the hypervariable locus DXS255 to DXS146, and also contains a chloride channel gene that is responsible for hereditary nephrolithiasis . We have used plasmid rescue, inverse PCR, and Alu-PCR to generate 20 novel markers from this region, 1 of which is polymorphic, and have positioned these relative to one another on the basis of YAC analysis . The order of previously known markers within our contigs, Xpter-OATL1-GATA-TFE3-SYP-DXS255146- Xcen, agrees with genomic pulsed-field maps of the region . In addition, we have constructed a rare-cutter restriction map for a 710-kb region of the DXS255-DXS146 contig and have identified three CPG islands . These contigs and new markers will provide a useful resource for more detailed analysis of Xp11.23-p11.22, a region implicated in several genetic diseases.

Genomics, 1995 Sep 20, 29(2), 478 - 89
YAC and cosmid contigs spanning the Batten disease (CLN3) region at 16p12.1-p11.2; Jarvela IE et al.; A yeast artificial chromosome (YAC) contig has been constructed in 16p12.1-p11.2 that encompasses three loci (D16S288, D16S299, and D16S298) closely linked to the gene causing Batten disease or juvenile-onset neuronal ceroid lipofuscinosis (CLN3) . The physical map has been ordered using 42 sequence tagged sites . Four genes, interleukin-4 receptor (IL4R), phenol-preferring phenol sulfotransferase (STP), monoamine-preferring phenol sulfotransferase (STM), and sialophorin (SPN), have been mapped to the YAC contig . A partial genomic restriction map has been constructed to confirm the order and distances between D16S298, predicted to be the locus closest to CLN3 . The overlapping genomic clones are a valuable resource for cloning the Batten gene (CLN3) and other genes in the region.

FEBS Lett, 1995 Sep 18, 372(1), 13 - 9
Isolation, characterization, and chromosomal location of a gene encoding the delta 1-pyrroline-5-carboxylate synthetase in Arabidopsis thaliana; Savoure A et al.; A full-length cDNA and the corresponding At-P5S gene encoding the first enzyme of the proline biosynthetic pathway, the delta 1-pyrroline-5-carboxylate (P5C) synthetase, were isolated in Arabidopsis thaliana . The At-P5S cDNA encodes a protein of 717 amino acids showing high identity with the P5C synthetase of Vigna aconitifolia . Strong homology is also found at the N-terminus to bacterial and yeast gamma-glutamyl kinase and at the C-terminus to bacterial gamma-glutamyl phosphate reductase . Putative ATP- and NAD(P)H-binding sites are suggested in the At-P5S protein . The transcribed region of the At-P5S gene is 4.8 kb long and contains 20 exons . Southern analysis suggests the presence of only one At-P5S gene in the A . thaliana genome mapped at the bottom of the chromosome two . Expression analysis of At-P5S in different organs reveals abundant At-P5S transcripts in mature flowering plant . Rapid induction of the At-P5S gene followed by accumulation of proline was observed in NaCl-treated seedlings suggesting that At-P5S is osmoregulated.

Science, 1995 Sep 15, 269(5230), 1580 - 3
A VAMP-binding protein from Aplysia required for neurotransmitter release; Skehel PA et al.; Before the fusion of synaptic vesicles with the plasma membrane, a protein complex is thought to form between VAMP--an integral membrane protein of the vesicle--and two proteins associated with the plasma membrane, SNAP-25 and syntaxin . The yeast two-hybrid interaction cloning system has now been used to identify additional proteins from Aplysia that interact directly with VAMP . A 33-kilodalton membrane protein, termed VAP-33 (VAMP-associated protein of 33 kilodaltons), was identified whose corresponding messenger RNA was detected only in the central nervous system and the gill of Aplysia . Presynaptic injection of antibodies specific for VAP-33 inhibited synaptic transmission, which suggests that VAP-33 is required for the exocytosis of neurotransmitter.

J Biol Chem, 1995 Sep 8, 270(36), 21307 - 11
Activation of transcription by recombinant upstream stimulatory factor 1 is mediated by a novel positive cofactor; Halle JP et al.; The transcription factor USF1 belongs to the family of basic helix-loop-helix proteins that are involved in the regulation of various important cellular processes . Here we characterized the factors involved in the activation of transcription by upstream stimulatory factor 1 (USF1) in a reconstituted class II gene transcription system . Activation of transcription by both wild type USF1 and a GAL-USF (amino acids 1-94 of the yeast activator protein GAL4 fused to amino acids 17-196 of USF) fusion protein required the presence of at least one positive cofactor . A novel positive cofactor (PC5) that functions specifically through the activation domain of USF1 was partially purified and biochemicaly distinguished from previously described positive cofactors . The mechanism by which PC5 mediates activation of transcription through USF1 was investigated in order-of-addition experiments . PC5 had to be present during binding of transcription factor (TF) IID to the TATA box to observe transcriptional activation . However, this event alone did not result in transcriptional activation, which also required the presence of the activator and of PC5 after binding of TFIID . Hence, PC5 may enter transcription during binding of TFIID to function in concert with the activator during subsequent steps in transcription.

Cell, 1995 Sep 8, 82(5), 815 - 21
The mei-41 gene of D . melanogaster is a structural and functional homolog of the human ataxia telangiectasia gene; Hari KL et al.; The D . melanogaster mei-41 gene is required for DNA repair, mitotic chromosome stability, and normal levels of meiotic recombination in oocytes . Here we show that the predicted mei-41 protein is similar in sequence to the ATM (ataxia telangiectasia) protein from humans and to the yeast rad3 and Mec1p proteins . There is also extensive functional overlap between mei-41 and ATM . Like ATM-deficient cells, mei-41 cells are exquisitely sensitive to ionizing radiation and display high levels of mitotic chromosome instability . We also demonstrate that mei-41 cells, like ATM-deficient cells, fail to show an irradiation-induced delay in the entry into mitosis that is characteristic of normal cells . Thus, the mei-41 gene of Drosophila may be considered to be a functional homolog of the human ATM gene.

J Biol Chem, 1995 Sep 8, 270(36), 21339 - 45
Universal minicircle sequence binding protein, a CCHC-type zinc finger protein that binds the universal minicircle sequence of trypanosomatids . Purification and characterization; Tzfati Y et al.; Replication of kinetoplast DNA minicircles of trypanosomatids initiates at a conserved 12-nucleotide sequence, termed the universal minicircle sequence (UMS, 5'-GGGGTTGGTGTA-3') . A single-stranded nucleic acid binding protein that binds specifically to this origin-associated sequence was purified to apparent homogeneity from Crithidia fasciculata cell extracts . This UMS-binding protein (UMSBP) is a dimer of 27.4 kDa with a 13.7-kDa protomer . UMSBP binds single-stranded DNA as well as single-stranded RNA but not double-stranded or four-stranded DNA structures . Stoichiometry analysis indicates the binding of UMSBP as a protein dimer to the UMS site . The five CCHC-type zinc finger motifs of UMSBP, predicted from its cDNA sequence, are similar to the CCHC motifs found in retroviral Gag polyproteins . The remarkable conservation of this motif in a family of proteins found in eukaryotic organisms from yeast and protozoa to mammals is discussed.

Biochem Pharmacol, 1995 Sep 7, 50(6), 787 - 96
Inhibition of 2,3-oxidosqualene cyclase and sterol biosynthesis by 10- and 19-azasqualene derivatives; Viola F et al.; The inhibition of 2,3-oxidosqualene-lanosterol cyclase (EC 5.4.99.7) (OSC) by new azasqualene derivatives, mimicking the proC-8 and proC-20 carbocationic high-energy intermediates of the cyclization of 2,3-oxidosqualene to lanosterol, was studied using pig liver microsomes, partially purified preparations of OSC, and yeast microsomes . The azasqualene derivatives tested were: 6E- and 6Z-10aza-10,11-dihydrosqualene-2,3-epoxide 17 and 18, 19-aza-18,19,22,23-tetrahydrosqualene-2,3-epoxide 19 and its corresponding N-oxide 20, and 19-aza-18,19,22,23-tetrahydrosqualene 21 . The compounds 17 and 19 (i.e . the derivatives bearing the 2,3-epoxide ring and the same geometrical configuration as the OSC substrate) were effective inhibitors, as shown by the Ki obtained using partially purified OSC: 2.67 microM and 2.14 microM, respectively . Compound 18, having an incorrect configuration and the 19-aza derivative 21, lacking the 2,3-epoxide ring, were poor inhibitors, with IC50 of 44 microM and 70 microM, respectively . Compound 21 was a competitive inhibitor of OSC, whereas 17 and 19 were noncompetitive inhibitors, and showed a biphasic time-dependent inactivation of OSC, their apparent binding constants being 250 microM and 213 microM, respectively . The inhibition of sterol biosynthesis was studied using human hepatoma HepG2 cells . The incorporation of {14C} acetate in the C27 sterols was reduced by 50% by 0.55 microM 17, 0.22 microM 19, and 0.45 microM 21, whereas 2 microM 18 did not affect sterol biosynthesis . In the presence of 17, 19 and 21, only the intermediate metabolites 2,3-oxidosqualene and 2,3,22,23-dioxidosqualene accumulated, demonstrating a very specific inhibition of OSC.

Biochem Biophys Res Commun, 1995 Sep 5, 214(1), 279 - 85
Novel isoform of beta 1 integrin expressed in skeletal and cardiac muscle; Zhidkova NI et al.; We describe a novel isoform of the beta 1 integrin subunit called beta 1D, which contains a unique cytoplasmic domain, and is expressed specifically in skeletal and cardiac muscle . The beta 1D isoform arises from splicing into the final transcript of an additional exon located between exons 6 and 7 . The nucleotide sequence of beta 1D predicts a cytoplasmic domain of 50 amino acids in which the last 21 amino acids of the beta 1A integrin isoform are replaced by a related sequence of 24 amino acids . A beta 1D-specific anti-peptide polyclonal antibody was prepared and immunoprecipitation of tissue extracts with subsequent immunoblotting showed expression of beta 1D isoform only in striated muscle cells.

FEBS Lett, 1995 Sep 4, 371(2), 119 - 22
The presence of H+ and Na(+)-translocating ATPases in Methanobacterium thermoautotrophicum and their possible function under alkaline conditions; Smigan P et al.; Two ATPases with different apparent molecular masses of approx . 500 kDa and 400 kDa were identified in the EDTA extract of the cell membranes of Methanobacterium thermoautotrophicum . Western blotting with polyclonal antiserum reactive with beta-subunit of mitochondrial ATPase from rat liver and yeast was used for further analysis of these ATPases . A strong crossreactivity with a single protein band with an apparent molecular weight of about 53 kDa (similar to beta-subunit of F-type ATPase from other sources) was found in protein extracts of whole cells of Methanobacterium thermoautotrophicum strains delta H and Marburg, as well as of Methanospirillum hungatei . This indicates the presence of F-type ATPase in methanogens . ATP synthesis driven by membrane potential which was generated by artificially-imposed delta pH in the presence of protonophorous uncoupler and sodium ions was stimulated by bafilomycin A1, an inhibitor of V- and A-type ATPases, as well as by harmaline, an inhibitor of Na+/H+ antiporter . These results indicate that cells of Methanobacterium thermoautotrophicum strain delta H contain the F-type ATP synthase which is Na(+)-translocating in addition to V- or A-type ATP synthase which is H(+)-translocating.

Genome Res, 1995 Sep, 5(2), 125 - 35
The organization of the human immunoglobulin lambda gene locus; Kawasaki K et al.; To elucidate the complex structure of the human immunoglobulin lambda gene locus, a 1020-kb contig was constructed using 184 cosmid clones and one bacterial artificial chromosome (BAC) clone . A high-resolution physical map of this contig revealed that the entire lambda gene locus is 911 kb in length . It contains seven constant region (C lambda) gene segments and 69 unique EcoRI-HindIII segments that hybridize to variable region gene (V lambda) probes . The VpreB gene, BCRL4, and gamma-glutamyl transpeptidase gene (GGT)-like sequences are also located within the lambda gene locus . Hybridization analysis suggested that the lambda gene locus has undergone extensive amplification events in evolution.

Somat Cell Mol Genet, 1995 Sep, 21(5), 345 - 9
Localization of the mouse lissencephaly-1 gene to mouse chromosome 11B3, in close proximity to D11Mit65; Peterfy M et al.; Lissencephaly is a human brain malformation manifested by a smooth cerebral surface and severe mental retardation . Some of the patients have been shown to have deletions in chromosome 17p13.3, and recently, LIS-1 has been proposed to be the disease-associated gene . We have now mapped the mouse homolog of LIS-1 to mouse chromosome 11B3 by using fluorescence in situ hybridization to metaphase chromosomes . The analysis of yeast artificial chromosome clones placed Lis-1 in close proximity to the microsatellite marker D11Mit65.

Acta Derm Venereol, 1995 Sep, 75(5), 361 - 3
Intracutaneous transport of orally administered fluconazole to the stratum corneum; Faergemann J et al.; Fluconazole administered at 150 mg/week for 1-5 weeks is effective orally against dermatophytes and yeast in stratum corneum . Clinical and mycological cure rates approach 90%, but the precise distribution of the drug within various layers of skin is uncertain . We administered fluconazole at 150 mg/week for 2 weeks to 5 volunteers . Distribution of fluconazole in biopsies of skin was imaged by energy dispersive analysis of X-rays (EDX) and transmission electron microscopy, and in cells by electron energy-loss spectroscopy (EELS) . Eight hours after a second dose, EDX showed fluconazole highest and homogeneously distributed in stratum corneum, lower in the rest of the epidermis, and lowest in dermis . The highest fluconazole levels detected by EELS were in cytoplasmic inclusions of sweat and sebaceous glands and less in keratinocytes and dermal collagen . We conclude that fluconazole delivered to stratum corneum by direct diffusion from capillaries and in sweat is also in all likelihood transported in sebum.

Acta Derm Venereol, 1995 Sep, 75(5), 357 - 60
Pityrosporum ovale extracts increase interleukin-4, interleukin-10 and IgE synthesis in patients with atopic eczema; Kroger S et al.; Evidence for a possible role of the lipophilic yeast Pityrosporum ovale in the pathophysiology of atopic eczema has been found both in laboratory and therapeutical studies . Positive type I prick test reactions to P . ovale correlate with the intensity of eczematous skin lesions in the head and neck regions of patients with atopic eczema . Furthermore, antifungal treatment has been shown to be helpful in atopic eczema . In the present study the effect of P . ovale on IgE synthesis and cytokine production (IL-2, IFN gamma, IL-4, IL-10) was investigated in patients with atopic eczema, in vitro . Eight patients with atopic eczema were studied; of these, 5 patients had specific IgE antibodies against P . ovale, as determined by fluoroimmunoassay (RAST) . The control group consisted of 5 healthy non-atopic, P . ovale IgE-antibody-negative volunteers . Freshly isolated peripheral blood mononuclear cells (PBMC) were incubated in the presence of different antigen concentrations (0.01, 0.1, 1.0, 10 micrograms/l x 10(6) cells) of P . ovale . IgE contents in the cell culture supematants were significantly elevated in RAST(+) patients with atopic eczema (p < 0.05), compared with RAST(-) atopic eczema patients and healthy volunteers . Coincubation of P . ovale-stimulated PBMC with IL-4 (50 U/l/ 1 x 10(6) cells) resulted in a significantly higher IgE synthesis only in the RAST(+) atopic eczema patients . Additionally, incubation of PBMC from RAST(+) patients with atopic eczema led to an elevated synthesis of the TH2 related cytokines IL-4 and IL-10 . Within the atopic eczema group, two subgroups differed markedly in their response to P . ovale antigen stimulation with a good correlation to the presence of specific IgE in serum and in vitro IL-4 and IL-10 production . The data support the assumption that P . ovale antigens might play a role in skin inflammation in at least a subgroup of patients with atopic eczema characterized by the presence of specific IgE antibodies to P . ovale.

Curr Genet, 1995 Sep, 28(4), 309 - 16
PkpA, a novel Phycomyces blakesleeanus serine/threonine protein kinase; Ruiz-Perez VL et al.; This work reports the cloning and sequencing of pkpA, a gene of the filamentous fungus Phycomyces blakesleeanus, whose expression seems to be coupled to vegetative growth . This gene encodes a putative serine/threonine-specific protein kinase, whose sequence is related to that of the yeast protein STE20, involved in pheromone-response pathways, and to a number of MAPK kinase proteins . However, detailed analysis of the kinase sequence suggests that PkpA is a novel serine/threonine protein kinase that probably participates as an intermediate in an intracellular system controlling nuclear proliferation in P . blakesleeanus.

Plant Cell, 1995 Sep, 7(9), 1459 - 71
Plant nuclear pore complex proteins are modified by novel oligosaccharides with terminal N-acetylglucosamine; Heese-Peck A et al.; Only a few nuclear pore complex (NPC) proteins, mainly in vertebrates and yeast but none in plants, have been well characterized . As an initial step to identify plant NPC proteins, we examined whether NPC proteins from tobacco are modified by N-acetylglucosamine (GlcNAc) . Using wheat germ agglutinin, a lectin that binds specifically to GlcNAc in plants, specific labeling was often found associated with or adjacent to NPCs . Nuclear proteins containing GlcNAc can be partially extracted by 0.5 M salt, as shown by a wheat germ agglutinin blot assay, and at least eight extracted proteins were modified by terminal GlcNAc, as determined by in vitro galactosyltransferase assays . Sugar analysis indicated that the plant glycans with terminal GlcNAc differ from the single O-linked GlcNAc of vertebrate NPC proteins in that they consist of oligosaccharides that are larger in size than five GlcNAc residues . Most of these appear to be bound to proteins via a hydroxyl group . This novel oligosaccharide modification may convey properties to the plant NPC that are different from those of vertebrate NPCs.

Anal Biochem, 1995 Sep 1, 230(1), 135 - 48
Tandem mass spectrometry and nuclear magnetic resonance spectroscopy studies of Candida bombicola sophorolipids and product formed on hydrolysis by cutinase; de Koster CG et al.; Natural mixtures of sophorolipids produced by the yeast Candida bombicola have been analyzed by fast atom bombardment (FAB)-MS and collision-induced dissociation (CID)-MS . Some pure components have been analysed by two-dimensional NMR spectroscopy . The presence of acidic, lactonic, and O-acetylated forms and the position of double bonds in the fatty acid part of these glycolipids can be easily inferred from positive and negative ion FAB-mass spectra . Details about position of O-acetylation can be obtained from CID mass spectra of {M+H}+ and {M-H}- ions and from the NMR spectra . Differences in CID fragmentation between protonated and sodiated molecular ions are discussed in detail . Enzymatic hydrolysis of 6',6"-di-O-acetyl sophorolipid lactone by cutinase from Fusarium solani results specifically in the removal of the 6'-O-acetyl group, whereas the 6"-O-acetyl and lactone group are resistant . This specificity is explained from a three-dimensional model of the sophorolipid generated on the basis of the short 1H,1H distances as inferred from the NMR (ROESY) spectra.

J Anim Sci, 1995 Sep, 73(9), 2721 - 6
Immune response, glucose metabolism, and performance of stressed feeder calves fed inorganic or organic chromium; Kegley EB et al.; One hundred twenty-five Angus crossbred steers (215 +/- 2 kg initial BW) were blocked by weight and assigned to pens . Pens were randomly assigned to treatment (six pens/treatment) . Treatments consisted of 1) control (no supplemental Cr), 2) CrCl3, 3) high-Cr yeast, or 4) Cr nicotinic acid complex . Chromium was added to provide .4 mg of supplemental Cr/kg of DM . Steers were fed diets containing 90% corn silage (DM basis) and 10% soybean meal-mineral-vitamin supplement . Steers were allowed to consume the diets on an ad libitum basis during the 56-d study . Performance was not affected by treatment . On d 52, steers supplemented with high-Cr yeast had a greater response to an intradermal injection of phytohemagglutin (PHA) for 8 h after injection than control steers (P < .10) or those supplemented with CrCl3 (P < .05) or Cr nicotinic acid (P < .05) . Peripheral lymphocytes from steers supplemented with Cr nicotinic acid had a greater (P < .05) blastogenic response to 12.5 micrograms PHA/mL than lymphocytes from steers supplemented with CrCl3 . After an i.v . infusion of glucose (.25 g of glucose/kg BW), plasma glucose tended (P < .11) to decrease at a faster rate from 15 to 45 min after infusion in steers fed Cr nicotinic acid . Steers supplemented with Cr nicotinic acid had greater (P < .05) serum insulin 15 and 30 min after infusion than those supplemented with CrCl3 and high-Cr yeast . Controls had lower serum insulin than those supplemented with Cr nicotinic acid 30 min after infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

J Androl, 1995 Sep-Oct, 16(5), 441 - 7
Selenium supplementation enhances the element concentration in blood and seminal fluid but does not change the spermatozoal quality characteristics in subfertile men; Iwanier K et al.; The objective of this study was to evaluate the effect of selenium (Se) supplementation on Se concentration and glutathione peroxidase (GSH-Px) activity in blood components and seminal fluid and on spermatozoal quality characteristics in subfertile men . Thirty-three men were supplemented for 12 weeks with 200 micrograms Se/day in the form of yeast-rich Se (group I, n = 16) or sodium selenite (group II, n = 17) . Blood samples and sperm were collected at the start of the study and after 2, 4, 8, and 12 weeks following Se supplementation . Se concentration in whole blood and plasma and GSH-Px activity in red cells and plasma increased significantly during the study, but in the group supplemented with yeast-Se the effect was more pronounced . Se concentration in seminal fluid also increased in both groups, but the effect of yeast-Se was markedly higher than that of selenite . In both groups statistically significant correlations were found between Se concentration in plasma and seminal fluid . GSH-Px activity in seminal fluid in the yeast-Se group increased significantly and reached a plateau after 2 weeks, whereas in the selenite group the activity did not change throughout the whole study period . Weak correlations between Se concentrations and GSH-Px activities in seminal fluid were seen, but only in the yeast-Se group were the relations statistically significant . The subjects in both groups showed no response in sperm count, motility, and morphology . In conclusion, we can ascertain that the supplementation of subfertile men with yeast-rich Se showed a more pronounced effect on Se concentrations and GSH-Px activities in blood components and seminal fluid than selenite did . Se supplementation did not improve the spermatozoal quality characteristics of sperm count, motility and, morphology.

Mycoses, 1995 Sep-Oct, 38(9-10), 373 - 6
Fatal fungaemia due to Sporothrix schenckii; Castrejon OV et al.; A clinical case is reported of a 78-year-old male with antecedents of diabetes and alcoholism who was hospitalized because he showed cutaneous lesions on the face and extremities suggesting cutaneous tuberculosis, but after a first histological study cutaneous leishmaniasis was erroneously diagnosed . Because of some unusual characteristics of the patient, the skin biopsies were carefully re-examined, as well as blood smears, which revealed elongated yeast form-like cells suggestive of Sporothrix schenckii . The diagnosis was confirmed when the fungus grew in mice and in Sabouraud cultures inoculated with blood samples from the patient . It is recommended that Sp . schenckii is included in the differential diagnosis of ulcerative cutaneous lesions in patients from Mexican humid areas.

Trans R Soc Trop Med Hyg, 1995 Sep-Oct, 89(5), 555 - 9
Acquired antibody levels to Plasmodium falciparum merozoite surface antigen 1 in residents of a highly endemic area of Papua New Guinea; al-Yaman F et al.; The prevalence and concentration of antibodies to a yeast-expressed N-terminal region (195A) and a baculo-virus-expressed C-terminal region (BVp42) of merozoite surface antigen 1 (MSA-1) were measured during a cross-sectional survey in the Wosera area of East Sepik Province, Papua New Guinea, in order to obtain baseline data on naturally acquired antibody response to this antigen in preparation for a vaccine trial . Overall, the seropositivity rate was 78% for 195A and 91% for BVp42 . Although antibody prevalence to both molecules increased with age, higher antibody prevalence rates were observed for BVp42 in all age groups studied . In children, significant positive associations were found between parasite prevalence and antibody prevalence for both regions of MSA-1 and between spleen rates and anti-BVp42 antibody prevalence . Concentration of antibody against both regions increased significantly with age, but was always higher for BVp42 . In children, antibody levels to both regions of MSA-1 were significantly higher in those infected (symptomatic and asymptomatic), while in adults no significant difference in antibody concentration was observed between those infected and those uninfected . However, enlarged spleens were associated with higher antibody concentration to both regions of MSA-1 in both children and adults . The C-terminal of MSA-1 appeared to be more recognized than the N-terminal, in terms of both antibody prevalence and concentration.

Vopr Med Khim, 1995 Sep-Oct, 41(5), 39 - 42
{Dynamics of luminol-dependent chemiluminescence in rat whole blood at different oxygen concentrations}; Prokof'ev VN et al.; Chemiluminescence analysis was used to examine the yeast cell-activation of rat whole blood neutrophilic leukocytes in rats after their pre-exposure to 3-hour hypoxia (9,000 m above sea level) and two-hour hyperoxia (0.3 MPa), as well as their resultant effects . The hypoxia was found to cause a significant (42%) reduction in the length of the latent period, suggesting that it activates the phagocytic activity of neutrophilic leukocytes . At the same time hyperoxia leads to a delayed response of competent cells to yeast cell stimulation . On subsequent exposure to hypoxia and hyperoxia, drastic inhibition of drastic cellular activation occurred, which manifested itself by a 62% increase in the length of the latent period and by a 38% rise in the appearance time of luminescence peak . Thus, the findings suggest that it is essential to bear in mind the fact that phagocytosis can decrease if oxygen is overdosed during hyperbaric oxygenation and in hypoxic states.

J Med Vet Mycol, 1995 Sep-Oct, 33(5), 355 - 8
A human isolate of Exophiala (Wangiella) dermatitidis forming a catenate synanamorph that links the genera Exophiala and Cladophialophora; de Hoog GS et al.; A strain of the black yeast Exophiala dermatitidis displayed a hydrophobic synanamorph consisting of acropetal chains of lemon-shaped conidia, morphologically similar to those of Cladophialophora bantiana . The occurrence of the two conidial types in a single strain suggests a taxonomic affinity between Exophiala and Cladophialophora and provides support to the supposition that Cladophialophora, a possible anamorph genus of Herpotrichiellaceae, is related to black yeasts rather than to Cladosporium, which has teleomorphs in the Mycosphaerellaceae.

J Med Vet Mycol, 1995 Sep-Oct, 33(5), 327 - 38
Phylogenetic relationships of human-pathogenic Cladosporium (Xylohypha) species inferred from partial LS rRNA sequences; Masclaux F et al.; The controversy about the appropriate taxonomic placement of agents of subcutaneous and systemic mycoses in either Cladosporium or Xylohypha, both genera characterized by conidia being produced in dry, acropetal chains, was addressed with partial sequencing of LS ribosomal RNA . Observation of catenate anamorphs in species of Capronia (Ascomycotina, Herpotrichiellaceae), a genus which also has anamorphs in Exophiala, suggested the possibility of a close interrelationship of all human-associated taxa . To test this hypothesis, partial sequences of 43 strains of Cladosporium/Xylohypha were analysed . Human-pathogenic and saprophytic Cladosporium species were found to be phylogenetically distinct from each other and, on the basis of known teleomorph relationships, were considered to be anamorphs of Herpotrichiellaceae and Mycosphaerellaceae, respectively . They should therefore be classified in different anamorph-genera; Cladosporium being restricted to plant-associated species . A relatively large proportion of the Herpotrichiellaceae is presumed to be animal-associated . The black yeast genus Exophiala was also confirmed to be of herpotrichiellaceous relationship . The genus Xylohypha is unrelated.

Hum Mol Genet, 1995 Sep, 4(9), 1509 - 18
cDNA selection from 10 Mb of chromosome 21 DNA: efficiency in transcriptional mapping and reflections of genome organization; Tassone F et al.; The technique of cDNA hybridization selection has been applied individually to 16 YAC clones mapping to various regions of the long arm of human chromosome 21 . YACs represented approximately 10 Mb of non-overlapping DNA, and cDNA sources included fetal brain, whole fetus, and adult testes, thymus, liver and spleen . Sequencing, Northern analysis, RT-PCR and cDNA library screenings have been used to identify and partially characterize a non-redundant set of novel genes . A preliminary analysis strategy of the selected cDNAs has proven rapid and effective for isolation of the more highly represented genes and is suitable for survey transcriptional mapping efforts . By scaling up to screen > 1000 cDNA fragments per 100 kb of YAC DNA, more rare transcripts are identified and lead to comprehensive gene maps . Strong regional variations in transcriptional activity were observed, with gene densities ranging from < 1/2000 kb to > 1/15 kb . This effort has produced a large number of new genes of potential relevance to Down Syndrome.

Genetics, 1995 Sep, 141(1), 263 - 70
Chaser (Csr), a new gene affecting larval foraging behavior in Drosophila melanogaster; Pereira HS et al.; Chaser (Csr) was uncovered in a gamma mutagenesis screen to identify genes that modify the larval foraging behavior of sitters to rovers . Rover larvae have significantly longer path lengths than sitters while foraging on a yeast and water paste . This difference is influenced by one major gene, foraging (for), which has two naturally occurring alleles, forR (rover) and fors (sitter) . In a mutagenesis screen for modifiers of for, we identified three lines with viable mutations on chromosome 3 that alter foraging behavior . Each of these mutations increased larval path lengths in fors/fors larvae in a dominant fashion, and were not separable by recombination . These mutations are therefore probably allelic and define a new gene that we have called Csr . Csr was genetically localized using the lethal-tagging technique . This technique resulted in seven lines with a significant decrease in larval path-length and recessive lethal mutations on chromosome 3 . We refer to these as reverted Csr (Csrrv) lines . Deficiencies that uncovered cytologically visible chromosome rearrangements in three of the seven reverted lines were used in a complementation analysis . In this way we mapped the lethal mutations in the Csrrv lines to cytological region 95F7-96A1 on the right arm of chromosome 3.

Mamm Genome, 1995 Sep, 6(9), 645 - 52
Molecular genetic analysis of the human sorbitol dehydrogenase gene; Carr IM et al.; The polyol pathway comprises the enzymes aldose reductase and sorbitol dehydrogenase, which convert glucose to sorbitol and sorbitol to fructose, respectively, particularly in hyperglycemic states . The accumulation and toxicity of sorbitol in specific tissues has been implicated in the development of microvascular problems in some diabetic patients . Inappropriate sorbitol accumulation in some patients may be the result of polymorphic variation in the human sorbitol dehydrogenase gene, causing reduced expression levels or enzymatic activity . We now describe the structure and expression profile of the human sorbitol dehydrogenase gene and identify a range of polymorphic variants that may be useful for co-segregation studies in diabetic patients with and without severe clinical complications from their disease.

Mamm Genome, 1995 Sep, 6(9), 617 - 22
Use of interspersed repetitive sequences-PCR products for cDNA selection; Villard L et al.; In order to increase the efficiency of cDNA selection approaches, we describe the use of interspersed repetitive sequences-PCR (IRS-PCR) products to isolate genes from large-insert genomic clones . IRS-PCR is conducted on total yeast DNA containing a YAC of interest so that there is no need to purify the starting genomic clone . This enables the production of large amounts of genomic substrate for cDNA selection and allows the use of unstable YAC clones . Moreover, the hybridization of the IRS-PCR product to the cDNA clones after selection introduces a positive selection step . We tested these PCR products from YACs for the presence of exons, using cDNAs originating from seven different genes . In each case, at least one exon was present in the IRS-PCR product . We have applied this strategy to four YAC clones originating from the human X Chromosome (Chr) . All the selected cDNAs, strongly positive with the IRS-PCR product, did indeed originate from a gene in the region covered by the YAC . In all cases, the previously known genes contained in the genomic clones have been isolated . In addition, we have isolated human genes that have already been described but not assigned to any chromosomal region.

Mol Biol Cell, 1995 Sep, 6(9), 1159 - 71
The C . elegans sex-determining gene fem-2 encodes a putative protein phosphatase; Pilgrim D et al.; The genetic and molecular analysis of genes involved in the regulation of sex determination in Caenorhabditis elegans suggests that the gene fem-2 plays an important role in regulating a pathway transducing a non-cell-autonomous signal to a nuclear transcription factor . The wild-type fem-2 gene was cloned by identifying sequences from the C . elegans physical map that could restore normal Fem-2 function to homozygous mutant fem-2 transgenic animals . cDNA sequences mapping to the minimal rescuing region correspond to an open reading frame with a sequence similar to protein phosphatase 2C enzymes from systems as diverse as yeast, humans, and plants, but the alignments suggest that FEM-2 falls into a separate class of proteins than the canonical homologues . Several fem-2 mutant alleles were sequenced, and the mutations are predicted to cause protein changes consistent with their observed phenotypes, such as missense mutations in conditional alleles, and a nonsense mutation in a predicted null allele . This is the first evidence implicating phosphorylation and/or dephosphorylation as a control mechanism in C . elegans sex determination.

Genomics, 1995 Sep 1, 29(1), 87 - 97
A 4-megabase YAC contig that spans the Langer-Giedion syndrome region on human chromosome 8q24.1: use in refining the location of the trichorhinophalangeal syndrome and multiple exostoses genes (TRPS1 and EXT1); Hou J et al.; We have constructed a physical map covering over 4 Mb of human chromosome 8q24.1 and used this map to refine the locations of the genes responsible for Langer-Giedion syndrome . The map is composed of overlapping YAC clones that were identified and ordered in relation to sequence tagged sites mapped to the Langer-Giedion chromosomal region on somatic cell hybrids . The minimal region of overlap of Langer-Giedion syndrome deletions, previously identified by analysis of 15 patients, was placed on the map by analysis of 2 patients whose deletions define the endpoints . The chromosome 8 breakpoint of a balanced t(8;9)(q24.11;q33.3) translocation from a patient with trichorhinophalangeal syndrome (TRPS I) was found to be located just within the proximal end of the minimal deletion region . A deletion of 8q24.11-q24.3 in a patient with multiple exostoses was found to overlap the distal end of the LGS deletion region, indicating that the EXT1 gene is distal to the TRPS1 gene and supporting the hypothesis that Langer-Giedion syndrome is due to loss of functional copies of both the TRPS1 and the EXT1 genes.

Genomics, 1995 Sep 1, 29(1), 261 - 5
Structure and organization of the human glucose phosphate isomerase gene (GPI); Walker JI et al.; Two overlapping yeast artificial chromosome clones containing the human glucose 6-phosphate isomerase gene (GPI) have been isolated . PCR and direct sequencing were used to determine the exon/intron structure of the gene . The gene spans in excess of 40 kb and consists of 18 exons ranging in size from 44 to 153 bp . All splice sites conform to the GT/AG rule.

Genomics, 1995 Sep 1, 29(1), 247 - 52
A high-resolution map of genes, microsatellite markers, and new dinucleotide repeats from UBE1 to the GATA locus in the region Xp11.23; Kwan SP et al.; Several new genes and markers have recently been identified on the proximal short arm of the human X chromosome in the area of Xp11.23 . We had previously generated a YAC contig in this region extending from UBE1 to the OATL1 locus . In this report two polymorphic dinucleotide repeats, DXS6949 and DXS6950, were isolated and characterized from the OATL1 locus . A panel of YAC deletion derivatives from the distal portion of the contig was used in conjunction with the rest of the YAC map to position the new microsatellites and order other markers localizing to this interval . The marker order was determined to be DXS1367-ZNF81-DXS6849-ZNF21-DXS6616-DXS 6950-DXS6949 . In the proximal region below OATL1, we have isolated a pair of YACs from the GATA locus, B1026 and C01160 . Mapping within these YACs indicates the orientation of DXS1126 and DXS1240, while a cosmid near the OATL1 region reveals the overlap between the YAC contigs from the two loci . This cosmid contains the gene responsible for Wiskott-Aldrich syndrome (WAS) and localizes the disease gene between OATL1 and GATA . These data enable the expansion of the present physical map of the X chromosome from UBE1 to the GATA locus, covering a large portion of the Xp11.23 region . Genetic cross-overs in Xp11.23 support the marker orientation and the position of WAS, contrary to previous reports . With the integration of both physical and genetic maps we have predicted the following marker order: Xpter-UBE1-SYN1/ARAF1/ TIMP1-DXS1367-ZNF81-DXS.6849-ZNF21-DXSy6616++ +-(OATL1, DXS6950-DXS6949)- WAS-(GATA, DXS1126)-DXS1240-Xcen.(ABSTRACT TRUNCATED AT 250 WORDS)

Genomics, 1995 Sep 1, 29(1), 170 - 8
A radiation hybrid map with 60 loci covering the entire short arm of chromosome 12; Raeymaekers P et al.; We present a high-resolution radiation hybrid map of the short arm of human chromosome 12 containing 60 loci, including 44 STSs within or closely associated with expressed sequences, 11 highly polymorphic markers, 2 anonymous sequences, 2 subtelomeric sequences, and 1 centromeric sequence . The 60 loci fell into 48 unique retention patterns, providing a comprehensive map covering the entire short arm of chromosome 12 with an average resolution of approximately 800 kb . Twenty-two unique positions were ordered in a 1000:1 framework map with an average resolution of 1.8 Mb . The proposed order is in good agreement with recently published genetic maps, high-resolution FISH maps, and YAC contigs . The noted inconsistencies involved neighboring loci permutations . Our observations further suggest the existence of chromosomal "hot spots" for breakage during irradiation . In three regions an usually high number of breaks was noted between neighboring loci compared to the physical distance derived from existing YAC contigs . Some of these hot spots seem to coincide with known chromosomal aberrations, of which at least two have been involved in the etiology of cancer.

Genomics, 1995 Sep 1, 29(1), 163 - 9
A YAC contig and an EST map in the pericentromeric region of chromosome 13 surrounding the loci for neurosensory nonsyndromic deafness (DFNB1 and DFNA3) and limb-girdle muscular dystrophy type 2C (LGMD2C); Guilford P et al.; Two forms of inherited childhood nonsyndromic deafness (DFNB1 and DFNA3) and a Duchenne-like form of progressive muscular dystrophy (LGMD2C) have been mapped to the pericentromeric region of chromosome 13 . To clone the genes responsible for these diseases we constructed a yeast artificial chromosome (YAC) contig spanning an 8-cM region between the polymorphic markers D13S175 and D13S221 . The contig comprises 24 sequence-tagged sites, among which 15 were newly obtained . This contig allowed us to order the polymorphic markers centromere-D13S175-D13S141-D13S143-D13S115-AF M128yc1-D13S292-D13S283-AFM323vh5- D13S221-telomere . Eight expressed sequence tags, previously assigned to 13q11-q12 (D13S182E, D13S183E, D13S502E, D13S504E, D13S505E, D13S837E, TUBA2, ATP1AL1), were localized on the YAC contig . YAC screening of a cDNA library derived from mouse cochlea allowed us to identify an alpha-tubulin gene (TUBA2) that was subsequently precisely mapped within the candidate region.

Genes Chromosomes Cancer, 1995 Sep, 14(1), 35 - 42
Balanced translocation in a neuroblastoma patient disrupts a cluster of small nuclear RNA U1 and tRNA genes in chromosomal band 1p36; van der Drift P et al.; Chromosomal band 1p36 probably harbours several neuroblastoma suppressor genes . A neuroblastoma patient has been described with a constitutional balanced translocation, t(1;17)(p36;q12-21) . Cytogenetically, no loss of chromosomal material was visible . The 1p36 translocation breakpoint could therefore have inactivated one allele of a tumour suppressor gene, thus predisposing the patient to develop neuroblastoma . We localized this breakpoint by pulsed field gel electrophoresis, analysis of yeast artificial chromosomes, and fluorescence in situ hybridization . Here we report that the breakpoint is within a large cluster of small nuclear RNA U1 (RNU1) and some tRNA genes (TRE, TRN) on chromosomal band 1p36 . The size of this cluster is over two megabases and it contains many other locally repeated sequences . Polyadenylated transcripts were identified for some of these sequences . In addition, the cluster is the target for integration of an adenovirus 5/SV40 hybrid virus . The translocation breakpoint maps distal of this viral integration site and proximal of marker PND.

Am J Pathol, 1995 Sep, 147(3), 580 - 5
Cytogenic and molecular analysis of an aggressive angiomyxoma; Kazmierczak B et al.; Aggressive angiomyxoma is a rare mesenchymal tumor occurring mainly in the vulvar region extending into the paravaginal and perirectal region . Histologically, this tumor is rich in vascular structures and in collagen fibers and is of myxoid appearance . Cytogenetic and molecular analysis was performed on a case of an aggressive angiomyxoma and revealed clonal karyotypic abnormalities . All 50 metaphases analyzed showed a translocation involving the chromosomal region 12q14-15 . Chromosomal aberrations involving the breakpoint region 12q14-15 are frequently seen in a variety of other mesenchymal tumors as uterine leiomyomas, lipomas, hamartomas of the lung, liposarcomas, or hemangiopericytomas . Therefore, this breakpoint region seems to be the most frequent chromosomal abnormality associated with the initiation of human mesenchymal neoplasms . To narrow down the breakpoint region on a molecular level in the cells of the angiomyxoma we performed FISH analysis with different cosmid clones originating from a yeast artificial chromosome and cosmid contig overspanning parts of the region 12q14-15 . We were able to narrow down the region to approximately 70-80 kb belonging to an area designated a multiple aberration region, because it also includes the breakpoints of leiomyomas, lipomas, and pleomorphic adenomas with 12q13-15 abnormalities . Our molecular and cytogenic data suggest that angiomyxomas or at least a subset of them molecularly belong to the benign group of mesenchymal tumors showing multiple aberration region involvement.

Arterioscler Thromb Vasc Biol, 1995 Sep, 15(9), 1424 - 31
Recombinant leech antiplatelet protein specifically blocks platelet deposition on collagen surfaces under flow conditions; van Zanten GH et al.; Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP) . This protein was cloned and expressed in yeast and blocks collagen-mediated platelet aggregation and the adhesion of platelets to collagen-coated plates under static conditions . In the current study we investigated the effect of rLAPP on platelet deposition to collagen and collagen-rich surfaces under flow conditions . rLAPP completely inhibited platelet adhesion on collagen types I, III, and IV with IC50 values of 70, 600, and 90 nmol/L, respectively (shear rate = 1600 s-1) . Approximately 10-fold more rLAPP was required to obtain a similar inhibition at a low shear rate of 375 s-1 . rLAPP caused a concentration-dependent inhibition of binding of 125I-von Willebrand factor (vWF) to collagen type III and was able to displace prebound vWF even after 24 hours . Since platelet adhesion at low shear rate is less dependent on vWF than at high shear rate, this property of rLAPP may explain why less rLAPP is needed at high shear rate than at low shear rate to produce the same effect . Platelet adhesion to collagen type VI was only partially inhibited by rLAPP (maximal 44% with 3 mumol/L rLAPP) . rLAPP also caused a pronounced inhibition of platelet deposition to cross sections of human atherosclerotic coronary arteries but had no effect on matrices of cultured human umbilical vein endothelial cells . rLAPP is a potent platelet adhesion inhibitor at high shear rate, which binds to collagen and works by inhibiting binding of vWF to collagen.

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