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 DN