Mol Cell Biol, 1995 Jan, 15(1), 94 - 101 The BN51 protein is a polymerase (Pol)-specific subunit of RNA Pol III which reveals a link between Pol III transcription and pre-rRNA processing; Jackson AJ et al.; The three eukaryotic nuclear RNA polymerase (Pol) contain common and unique subunits . Cloning of the unique Pol III subunit genes in yeast cells has revealed a potential homolog in the mammalian system, the BN51 gene . The human BN51 gene was originally isolated as a suppressor of a temperature-sensitive cell cycle mutant of BHK cells (tsBN51) . Although tsBN51 cells have a marked decrease in RNA Pol III activity at the nonpermissive temperature, direct biochemical evidence for the BN51 protein being a human Pol III subunit was lacking . Using antibodies directed against the BN51 protein, we show the following: (i) the BN51 protein copurifies with Pol III activity, (ii) Pol III activity can be specifically immunoprecipitated from HeLa nuclear extracts, and (iii) the immunopurified BN51 complex is active in restoring both nonspecific and promoter-specific Pol III activity . Our findings provide direct biochemical evidence for BN51 being a Pol III-specific subunit . Despite the fact that BN51 is not a subunit of Pol I, the production of mature Pol I transcripts is inhibited in tsBN51 cells at the nonpermissive temperature . tsBN51 cells appear defective in processing the 32S precursor rRNA into mature 5.8S and 28S rRNA at the nonpermissive temperature . We surmise that ribosome assembly has halted because of the loss of Pol III transcripts . Thus, there is regulation of the synthesis of mature Pol I transcripts by a posttranscriptional mechanism based on the availability of Pol III transcripts. Mol Cell Biol, 1995 Jan, 15(1), 186 - 97 Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1; Richard S et al.; src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains . Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood . To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain . By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein . Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn . The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase . As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions . Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo . Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules. Cytogenet Cell Genet, 1995, 70(3-4), 183 - 5 Determination of the gene order of the three loci CD2, NGFB, and NRAS at human chromosome band 1p13 and refinement of their localisation at the subband level by fluorescence in situ hybridisation; Mitchell EL et al.; The three loci NRAS, NGFB, and CD2 map to human chromosome band 1p13 . Using fluorescence in situ hybridisation (FISH) to simultaneously DAPI-banded metaphase chromosomes, we have further refined the localisation of these three genes to specific subbands . NRAS localises to subband 1p13.2 and CD2 and NGFB to 1p13.1 . Also, with the use of multicolour FISH, we have determined the order and orientation of the three loci in relation to the centromere . The order is cen-CD2-NGFB-NRAS. Int J Immunopharmacol, 1995 Jan, 17(1), 49 - 53 In vivo and in vitro effects of macrophage colony-stimulating factor (M-CSF) on bronchoalveolar macrophages for antihistoplasmal activity; Khemani S et al.; The in vivo and in vitro effects of M-CSF on bronchoalveolar macrophages (BAM) activity against the intracellular fungal pathogen Histoplasma capsulatum (Hc) were studied . Three days after a single subcutaneous (s.c.) dose of M-CSF (2.5 mg/kg), enhanced ex vivo antifungal activity of BAM was measured . BAM from M-CSF-treated CD-1 mice significantly (P < 0.01) inhibited the intracellular multiplication of Hc yeast cells in 20 h assays compared to BAM from control mice . This effect was not observed at days 1, 7, 11 or 21 post-treatment . A dose of 5 mg/kg s.c., but not 1 mg/kg, induced similar antifungal activity in BAM by day 3 . Peritoneal macrophages (PM) from M-CSF-treated mice did not have enhanced antifungal activity at days and doses tested . BAM could also be activated for antihistoplasmal activity by M-CSF in vitro . M-CSF at 10,000 U/ml for 24 h or 5000 U/ml for 48 h induced significant (P < 0.01) inhibition of intracellular multiplication of Hc . Interferon-gamma (IFN) plus lipopolysaccharide (LPS) activated BAM and PM in vitro to inhibit intracellular multiplication of Hc (P < 0.001); the antihistoplasmal activity was completely inhibited by NG-monomethyl L-arginine (N-MMA), indicating that an L-arginine-dependent nitric oxide-producing mechanism was operative . N-MMA could not inhibit the antihistoplasmal activity of BAM or PM activated by M-CSF in vitro . The mechanism by which M-CSF-activated macrophages inhibit intracellular multiplication of Hc remains to be determined. Biol Trace Elem Res, 1995 Jan-Mar, 47(1-3), 201 - 7 Low selenium status in alcoholic cirrhosis is correlated with aminopyrine breath test . Preliminary effects of selenium supplementation; Van Gossum A et al.; The relationship among impaired selenium status, lipid peroxidation, and liver function was examined in 19 hospitalized patients with severe alcoholic cirrhosis . Plasma selenium was found to be significantly lower (mean +/- SD: 54 +/- 13 micrograms/L) than in healthy controls (83 +/- 11 micrograms/L) and plasma malondialdehyde, assessed as thiobarbituric acid reactants, which reflects lipid peroxidation, was increased (2.0 +/- 1.2 mumol/L vs < 1.2 mumol/L in controls) . The mean 14C aminopyrine breath test, an indicator of liver function, was lower than normal (2.7 +/- 1.9 vs 6.3 +/- 0.9% in controls) and found to be significantly correlated with plasma selenium (r = 0.59, p < 0.05) . A prospective, randomized selenium supplementation trial was conducted in a group of 16 patients who received either daily 100 micrograms selenium as enriched yeast during 4 mo or a placebo . Among the 10 patients who completed the study, plasma selenium significantly increased in the supplemented group (n = 4; before: 58 +/- 10 micrograms/L, and after 101 +/- 12 micrograms/L, p < 0.01) contrary to the placebo group (n = 6, before: 47 +/- 10 micrograms/L, after: 57 +/- 9 micrograms/L, n.s.) . 14C aminopyrine breath test improved in three out of four selenium-supplemented patients and in three out of six placebo patients, but the small number of patients did not allow statistical evaluation . These results demonstrate that low selenium status in alcoholic cirrhosis is correlated to liver function and could be improved by supplementation. J Protein Chem, 1995 Jan, 14(1), 19 - 25 Conserved nonplanar heme distortions in cytochromes c; Hobbs JD et al.; A nonplanar distortion of the heme of c-type cytochromes is conserved in the proteins isolated from diverse species based upon a comprehensive analysis of available high-resolution X-ray crystal structures . This distortion is induced through the cysteine thioether linkages between the porphyrin pyrrole groups and the polypeptide and results in an asymmetric pyrrole distortion . This asymmetry in the heme distortion is also conserved . For other heme proteins which lack these covalent bonds, nearly planar porphyrins are observed . Resonance Raman evidence indicates that nonplanar distortion of porphyrins containing metals, like iron, with large core sizes (> or = 2.00 A) is energetically unfavorable and can occur only in the presence of significant environmental perturbations . Further, energy minimization and dynamics calculations on the ferric form of yeast iso-1-cytochrome c, starting from the crystallographic coordinates and using a molecular mechanics force field which accurately reproduces nonplanar distortions in metalloporphyrins, suggest that this distortion is indeed maintained by the protein tertiary structure . It is proposed that this protein-linked heme distortion modulates electron transfer function through modification of redox potentials of the porphyrin ring and the protein binding properties of c-type cytochromes. J Biochem (Tokyo), 1995 Jan, 117(1), 163 - 8 Isolation and identification of adenosine triphosphoribosyl nicotinamide adenine dinucleotidephosphate from Azotobacter vinelandii; Imai T et al.; A novel type of pyridine nucleotide, containing two adenosine triphosphate ribose residues rather than one, was isolated from Azotobacter vinelandii strain O . The nucleotide was shown to be 2"- or 3"-(2'-phosphoadenosine-5'-diphosphoribosyl)nicotinamide adenine dinucleotide phosphate, in which 2'-phospho-5'-diphosphoadenosylribose was glycosidically linked to the NADP at position 2' or 3' of the nicotinamide mononucleotide moiety . The ATPribosylNADP did not show coenzyme activity for yeast glucose 6-phosphate dehydrogenase, nor was it cleaved by Neurospora crassa NAD(P) glycohydrolase, indicating that the biological properties conferred on the beta-NADP molecule were largely modified by the attachment of the ATP-ribose group. Genomics, 1995 Jan 1, 25(1), 59 - 65 A 500-kilobase region containing the tuberous sclerosis locus (TSC1) in a 1.7-megabase YAC and cosmid contig; Murrell J et al.; A complete overlapping clone map of a 1.7-Mb region from DBH to D9S67 that includes the TSC1 candidate region has been constructed . The map includes YAC and cosmid clones, contains STS approximately every 50 kb on average, and establishes the order of five previously unordered loci . The overall physical length of this segment of chromosome 9q34 (1.7 Mb) is significantly less than expected compared to its estimated genetic length (approximately 10 cM) . Consequently, the physical length of the TSC1 candidate region is substantially less than predicted by a genetic distance of approximately 2 cM. Genomics, 1995 Jan 1, 25(1), 304 - 8 Physical analysis of the tuberous sclerosis region in 9q34; Zhou CY et al.; We report the construction of a physical map based on cloned DNA within the candidate region for the tuberous sclerosis complex (TSC1) gene on chromosome 9q34, between the markers D9S149 and D9S66 . The DNA clones form three contigs consisting of 7 YACs, bridged by P1 and cosmid clones, and cover more than 950 kb of 9q34 . Despite intensive screening of all available libraries, two gaps remain . A detailed physical map of much of this region was derived, and restriction mapping of the YAC, P1, and cosmid clones reveals novel CpG islands in this region . This set of genomic clones provides a resource for characterizing candidates for the TSC1 gene, guided by the location of CpG islands. Genomics, 1995 Jan 1, 25(1), 295 - 7 A physical map encompassing GP2B, EPB3, D17S183, D17S78, D17S1183, and D17S1184; Miki Y et al.; The q21 region of chromosome 17 contains the gene BRCA1, which is involved in familial early-onset breast and ovarian cancers . A physical map of a region that extends from a distal boundary of the BRCA1 region, D17S78, to GP2B has been constructed . The map consists of 30 STSs, including 2 new short tandem repeat polymorphic markers . The contig is composed of a mixture of 7 YACs, 5 P1 plasmids, and 14 cosmids and was ordered by STS-content mapping. Genomics, 1995 Jan 1, 25(1), 264 - 73 A YAC-, P1-, and cosmid-based physical map of the BRCA1 region on chromosome 17q21; Couch FJ et al.; A familial early-onset breast cancer gene (BRCA1) has been localized to chromosome 17q21 . To characterize this region and to aid in the identification of the BRCA1 gene, a physical map of a region of 1.0-1.5 Mb between the EDH17B1 and the PPY loci on chromosome 17q21 was generated . The physical map is composed of a yeast artificial chromosome (YAC) and P1 phage contig with one gap . The majority of the interval has also been converted to a cosmid contig . Twenty-three PCR-based sequence-tagged sites (STSs) were mapped to these contigs, thereby confirming the order and overlap of individual clones . This complex physical map of the BRCA1 region was used to isolate genes by a number of gene identification techniques and to generate transcript maps of the region, as presented in the three accompanying manuscripts of Brody et al . (1995), Osborne-Lawrence et al . (1995), and Friedman et al . (1995). Genomics, 1995 Jan 1, 25(1), 256 - 63 22 genes from chromosome 17q21: cloning, sequencing, and characterization of mutations in breast cancer families and tumors; Friedman LS et al.; In our effort to identify BRCA1, 22 genes were cloned from a 1-Mb region of chromosome 17q21 defined by meiotic recombinants in families with inherited breast and/or ovarian cancer . Subsequent discovery of another meiotic recombinant narrowed the region to approximately 650 kb . Genes were cloned from fibroblast and ovarian cDNA libraries by direct screening with YACs and cosmids . The more than 400 cDNA clones so identified were mapped to cosmids, YACs, and P1 clones and to a chromosome 17 somatic panel informative for the BRCA1 region . Clones that mapped back to the region were hybridized to each other and consolidated into clusters reflecting 22 genes . Ten genes were known human genes, 5 were human homologs of known genes, and 7 were novel . Each gene was sequenced, compared to genes in the databases to find homologies, and analyzed for mutations in BRCA1-linked families and tumors . Eight mutations were found in tumors or families and not in controls . In the gene encoding alpha-N-acetylglucosaminidase, approximately 100 kb proximal to the 650-kb linked region, somatic nonsense, missense, and splice junction mutations occurred in 3 breast tumors, but not in these patients' germline DNA nor in controls . In an ets-related oncogene in the linked region, a missense mutation cosegregated with breast cancer in one family and was not observed in controls . In a human homolog of a yeast pre-mRNA splicing factor, 3 different mutations cosegregated with breast cancer in 3 families and were not observed in controls . In these and the other genes in the region, 36 polymorphic variants were observed in both cases and controls. Genomics, 1995 Jan 1, 25(1), 248 - 55 Direct selection of expressed sequences within a 1-Mb region flanking BRCA1 on human chromosome 17q21; Osborne-Lawrence S et al.; Direct selection of genes within the interval of chromosome 17q21 containing BRCA1 was performed . YAC and cosmid contigs spanning the BRCA1 region were used to select cDNA clones from pools of cDNAs derived from human placenta, HeLa cells, activated T cells, and fetal head . A minimum set of 48 fragments of nonoverlapping cDNAs that unequivocally mapped within a 1-Mb region was identified, although it is not yet known how many of these are derived from the same transcript . DNA sequence analyses revealed that 4 of these cDNAs were derived from known genes (EDH17B2, glucose-6-phosphatase, IAI.3B, and E1AF), 1 is a member of a previously described gene family (HMG-17), and 7 share substantial identity with previously described genes from human or other species . The remainder showed no significant homology to known genes . Limited PCR-based expression profiles of a set of 13 of the genes were performed, and all gave positive results with at least some cDNA sources supporting the contention that they truly represent transcribed sequences . A comparison between genes obtained from this region by direct selection with those obtained by direct screening or exon trapping (see accompanying papers, this issue) revealed that over 90% of the genes identified by exon trapping were represented in the selected material and that at least two additional genes that appear to represent low abundance transcripts with restricted expression profiles were identified by selection but not by other means. Genomics, 1995 Jan 1, 25(1), 238 - 47 Construction of a transcription map surrounding the BRCA1 locus of human chromosome 17; Brody LC et al.; We have used a combination of methods (exon amplification, direct selection, direct screening, evolutionary conservation, island rescue-PCR, and direct sequence analysis) to survey approximately 600 kb of genomic DNA surrounding the BRCA1 gene for transcribed sequences . We have cloned a set of fragments representing at least 26 genes . The DNA sequence of these clones reveals that 5 are previously cloned genes; the precise chromosomal location of 2 was previously unknown, and 3 have been cloned and mapped by others to this interval . Three other genes, including BRCA1 itself, have recently been mapped independently to this region . Sequences from 11 genes are similar but not identical matches to known genes; 5 of these appear to be the human homologues of genes cloned from other species . Another 7 genes have no similarity with known genes . In addition, 39 putative exons and 14 expressed sequence tags have been identified and mapped to individual cosmids . This transcript map provides a detailed description of gene organization for this region of the genome. Genomics, 1995 Jan 1, 25(1), 19 - 28 YAC contigs covering an 8-megabase region of 3p deleted in the small-cell lung cancer cell line U2020; Todd S et al.; Somatic deletions of chromosome 3p occur at high frequencies in cancers of kidney, breast, cervix, head and neck, nasopharynx, and lung . The frequency of 3p deletion in lung cancer approaches 100% among small cell lesions and 70 to 80% in non-small cell lesions . This evidence strongly implies that one or more tumor suppressor genes of potentially widespread significance reside within the deleted region(s) . Precise definition of the deleted target region(s) has been difficult due to the extensive area(s) lost and use of markers with low informativeness . However, improved definition remains essential to permit isolation of putative tumor suppressor genes from 3p . The identification of several small, homozygous 3p deletions in lung cancer cell lines has provided a critical resource that will assist this search . The U2020 cell line contains a small homozygous deletion that maps to a very proximal region of 3p and includes the marker D3S3 . We previously identified a subset of DNA markers located within the deleted region and determined their relative order by pulsed-field gel mapping studies . In the present report, we describe the development of YAC contigs that span the majority of the deleted region and link up to flanking markers on both sides . The centromere proximal portion of the contig crosses the breakpoint from an X;3 translocation located within 3p12 providing both location and orientation to the map . PCR-based (CA)n microsatellite polymorphisms have been localized within and flanking the deletion region . These markers should greatly facilitate loss-of-heterozygosity studies of this region in human cancer . The contig provides a direct means for isolation of putative tumor suppressor genes from this segment of 3p. Genomics, 1995 Jan 1, 25(1), 184 - 91 A boundary of long-range G + C% mosaic domains in the human MHC locus: pseudoautosomal boundary-like sequence exists near the boundary; Fukagawa T et al.; The human genome is composed of long-range G+C% (GC%) mosaic structures related to chromosome bands . We found the human MHC locus to be an example of megabase-level GC% mosaic structures and predicted a possible boundary of the megabase-level domains within an undercharacterized 450-kb region harboring the junction of MHC classes II and III . Chromosome walking of the 450-kb region and base-compositional analysis precisely located the boundary of the mosaic domains, disclosing a sharp GC% transition . Near the transition point there was a 20-kb dense Alu cluster, a 30-kb dense LINE-1 cluster, and a sequence highly homologous with the pseudoautosomal boundary of the short arms of human sex chromosomes (PAB1X and PAB1Y); PAB1X and PAB1Y are the interface between sex-specific and pseudoautosomal regions . Many PAB1XY-like sequences (PABLs) were detected by hybridization against genomic DNA, and the new sequences defined the complete form of PABLs to be about 650 nt. Protein Sci, 1995 Jan, 4(1), 21 - 8 A recombinant human hemoglobin with asparagine-102(beta) substituted by alanine has a limiting low oxygen affinity, reduced marginally by chloride; Yanase H et al.; A recombinant (r) mutant hemoglobin (Hb) with Asn-102(beta) replaced by an Ala (N102A(beta)) has been prepared by PCR amplification of a mutagenic DNA fragment and expression of the recombinant protein in yeast . The side chain of Asn-102(beta) is part of an important region of the alpha 1 beta 2 interface that undergoes large structural changes in the transition between the deoxy and oxy conformations . Three natural mutant Hbs with neutral substitutions of Thr, Ser, or Tyr at this site have low oxygen affinities because a hydrogen bond between Asn-102(beta) and Asp-94(alpha) in normal HbA was considered to be absent in these mutants, thereby destabilizing the oxy conformation in favor of the deoxy conformation . This proposal has been tested by expression of an rHb containing alanine at position 102(beta); alanine was chosen because its methyl side chain cannot participate in hydrogen bond formation, yet it is small enough not to disrupt the subunit interface . The nature of the desired replacement was established by sequencing the entire mutated beta-globin gene as well as the tryptic peptide containing the substitution . Further characterization by SDS-PAGE, isoelectric focusing, HPLC analysis, mass spectrometry, amino acid analysis, and sequencing of the mutant tryptic peptide confirmed the purity of the rHb . Its oxygen binding curve (2.4 mM in heme) in the absence of chloride showed that it had a very low oxygen affinity with a P50 of 42 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS) Eur J Hum Genet, 1995, 3(1), 56 - 60 Mapping of the spinal muscular atrophy (SMA) gene to a 750-kb interval flanked by two new microsatellites; Wirth B et al.; The gene for autosomal recessive proximal spinal muscular atrophy (SMA) has recently been mapped between D5S629 and D5S557 . We report here a new single-locus microsatellite A31 (D5S823) and two multicopy microsatellites 97T-CA and 95/23-CA . The marker A31 maps to the region of overlap between YACs y116, y55 and y122, distal to D5S629; 97T-CA originates from a cosmid corresponding to the STS 97T, localized distally to A31, while 95/23-CA derives from a cosmid corresponding to the STS 97U, localized proximally to D5S557 . We tested all our key recombinant families with these markers . In one type I/II SMA family, a recombinant was found that placed the SMA locus distal to D5S823 . Homozygosity mapping in a consanguineous type I SMA family indicates that the SMA gene lies proximal to 95/23-CA . Thus, the two new markers, A31 and 95/23-CA further refine the SMA gene to an approximately 750-kb interval. Planta, 1995, 195(3), 396 - 402 The general mitochondrial processing peptidase from wheat is integrated into the cytochrome bc1-complex of the respiratory chain; Braun HP et al.; The bc1-complex (EC 1.10.2.2.) from Triticum aestivum L . was purified by cytochrome-c affinity chromatography and gel filtration using either etiolated seedlings or wheat-germ extract as starting material . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated enzyme revealed ten bands, which were analysed by immunoblotting and direct amino-acid sequencing . The enzyme from wheat is the first bc1-complex that is reported to contain four core proteins (55.5, 55.0, 51.5 and 51.0 kDa) . In addition, the wheat bc1-complex comprises cytochrome b (35 kDa), cytochrome c1 (33 kDa) the "Rieske" iron-sulphur protein (25 kDa) and three small subunits < 15 kDa . This composition differs from the one reported in fungi, mammals and potato . Partial sequence determination of the large subunits suggests that the 55.5- and 55.0-kDa-proteins represent the beta-subunit of the general mitochondrial processing peptidase, and the 51.5- and 51.0-kDa proteins the alpha-subunit of this enzyme . The bc1-complex from wheat efficiently processes mitochondrial precursor proteins as shown in an in-vitro processing assay . In control experiments the isolated bc1-complexes from potato, yeast, Neurospora and beef, all purified by the same isolation procedure, were also tested for processing activity . Only the protein complexes from plants contain the general mitochondrial processing peptidase . The composition of the wheat bc1-complex sheds new light on the co-evolution of the processing peptidase and the middle segment of the respiratory chain. J Mol Cell Cardiol, 1995 Jan, 27(1), 589 - 97 Characterization of a novel transcript of retroviral origin expressed in rat heart and liver; Martin XJ et al.; Eleven cDNA clones of retroviral origin (SORO) have been isolated and characterized from a rat cardiac cDNA library that are highly homologous to the RAL-element family of rat retroviral endogenous sequences . SORO-1, the largest of the clones isolated at approximately 3.5 kb, contains a characteristic long terminal repeat (LTR) that is unique to previously identified retroposons . Its LTR contains elements homologous to the CAAT box, a TATA box, a core enhancer sequence, and potential binding sites for GATA . SORO-1 hybridizes to a mRNA of approximately 7 kb, that is present both in rat heart and liver . This transcript was not, however, detected in other tissues (fast and slow skeletal muscles, brain, kidney, testis, lung, intestine, spleen) or from any other species (man, monkey, mouse, dog, rabbit, chicken, cow, yeast) examined . During postnatal cardiac development, the expression of the transcript appears to be down-regulated and is present at levels 10-fold greater in neonates than in adults . The function of this retroposon that is expressed in rat heart and liver has not yet been determined, but the sequence analysis of its LTR and its expression pattern suggest that it may be regulated by specific trans-acting factors that are present in both rat heart and liver. Hum Exp Toxicol, 1995 Jan, 14(1), 55 - 60 Inhibition of aflatoxin B1-induced cell injury by selenium: an in vitro study; Shi CY et al.; Dietary selenium is an essential trace element in human nutrition . Selenium has been shown in animal studies to inhibit aflatoxin hepatocarcinogenesis . However, the cellular mechanism responsible for the inhibition has not been thoroughly studied . This study examines the effect of two selenium compounds, namely, sodium selenite and selenium-enriched yeast extract (SeY), on the cytotoxicity, DNA-binding and mutagenicity of aflatoxin B1 (AFB1) in cultured Chinese hamster ovary (CHO) cells . CHO cells, after treatment with 2 micrograms ml-1 selenite or 80 micrograms ml-1 SeY, exhibited increased resistance to AFB1-induced cell killing . At a concentration of 50 micrograms ml-1 AFB1, cell survival, measured by the clonogenicity assay, was increased by 21- and 10-fold in selenite- and SeY-treated cells, respectively . However, selenium treatment did not appear to affect AFB1-DNA binding . Similarly, no effect was observed on AFB1 mutagenicity, as determined by the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutation assay . The results showed that selenium could effectively protect cells from AFB1 cytotoxicity in cultured cells but had no effect on AFB1-DNA adduct formation or mutagenesis . It is suggested that there are multiple pathways of AFB1 toxicity and that selenium can modulate AFB1-induced cell killing independent of its genotoxicity. Mol Microbiol, 1995 Jan, 15(2), 235 - 45 Missense mutations in the catalase-peroxidase gene, katG, are associated with isoniazid resistance in Mycobacterium tuberculosis; Heym B et al.; The toxicity of the powerful anti-tuberculosis drug isoniazid (INH) is believed to be mediated by the haem-containing enzyme catalase-peroxidase, encoded by the katG gene of Mycobacterium tuberculosis . Compelling evidence for this was obtained by studying a panel of INH-resistant clinical isolates using a novel strategy based on the polymerase chain reaction and single-strand-conformation polymorphism analysis (PCR-SSCP) to detect mutations in katG . In most cases INH resistance was associated with missense mutations while in a small number of strains the gene had been completely, or partially, deleted . The missense mutations fell into two groups, the larger of which contained several independent mutations that affected the N-terminal peroxidase domain of the protein, resulting in the production of a catalase peroxidase with strongly reduced enzyme activity and increased heat liability . The effects of these substitutions could be interpreted by means of molecular modelling using the crystal structure of the related enzyme cytochrome c peroxidase from yeast as a template . The second group comprises a frequently occurring amino acid substitution and a single mutation that are both located in the C-terminal domain but do not noticeably alter either enzyme activity or heat stability. Muscle Nerve, 1995, 2, S14 - 8 Fish mapping of 250 cosmid and 26 YAC clones to chromosome 4 with special emphasis on the FSHD region at 4q35; Wijmenga C et al.; Facioscapulohumeral muscular dystrophy (FSHD) is located on chromosome 4q35, close to the telomere . FSHD patients carry deletions within a cluster of tandemly repeated DNA . Although expression of a functional FSHD gene will be altered in patients, the sequence itself may be unaffected by this deletion . Hence, the FSHD gene could lie outside of the deleted region . This study employs fluorescent in situ hybridization using chromosome 4-specific cosmid and YAC clones to rapidly saturate chromosome 4 with new markers . Some 250 cosmids and 26 YACs were regionally mapped, of which 5 YACs and 55 cosmids mapped to the distal portion of 4q . Only one of these clones (D4S1454) mapped telomerically to a translocation breakpoint specified by D4S187 . Using two-color interphase mapping, the following marker order was obtained: Cen-D4S187-D4S1454-HSPCAL2-D4S163-D4S139-+ ++D4F35S1 . Absence of additional markers mapping distal to D4F35S1 indicates that the linkage group containing the FSHD gene lies extremely close to the 4q telomere. Mamm Genome, 1995 Jan, 6(1), 37 - 41 Construction of a porcine YAC library and mapping of the cardiac muscle ryanodine receptor gene to chromosome 14q22-q23; Leeb T et al.; Large-scale physical mapping of the porcine genome has been limited because up to now no suitable genomic libraries for this purpose have been available . Therefore, we have constructed a yeast artificial chromosome (YAC) library from porcine lymphocytes . The library was cloned in the amplifiable vector pCGS966 . A total of 10080 YAC clones was obtained and has been ordered into 105 96-well microtiter plates . An average insert size of 300 kb was calculated from the analysis of 78 randomly selected clones, giving a one-fold coverage of the porcine genome . To analyze the complexity, we have screened the library for five different genes and isolated four different clones containing parts of three of these genes . One YAC clone harboring parts of the porcine cardiac muscle ryanodine receptor (RYR2) gene allowed us to assign this locus to Chromosome (Chr) 14q22-q23 . The data were confirmed by PCR analysis of a rodent-porcine hybrid cell panel. J Mol Evol, 1995 Jan, 40(1), 13 - 24 Gypsy/Ty3-class retrotransposons integrated in the DNA of herring, tunicate, and echinoderms; Britten RJ et al.; Eight new examples of retrotransposons of the Gypsy/Ty3 class have been identified in marine species . A 525-nt pol gene-coding region was amplified using degenerate primers from highly conserved regions and has extended the range of recognition of Gypsy/Ty3 far beyond those previously known . The following matrix shows the percentage AA divergence of the translations of this segment of the pol gene coding region . {table: see text} The underlines separate three groups of retrotransposons that can be recognized on the basis of this amino acid sequence . The new upper group shows surprising amino acid sequence similarity among members from the DNA of herring, sea urchin, starfish, and a tunicate . For example, the herring element differs by only 41% from the Ciona element and 46% from the sea urchin element . The group between the lines includes members close to previously known elements (marked by asterisks) and has so far been found only in sea urchins . The two upper groups differ from each other by 55-60% and yet members of both groups (e.g., Spr1 and Spr2) are integrated into the DNA of one species--S . purpuratus . Below the lower underline is listed the only known representative of a very distant group, which occurs in starfish DNA . In spite of large divergence, amino acid sequence comparisons indicate that all of the elements shown in the array are members of the LTR-containing class of retrotransposons that includes Gypsy of Drosophila and Ty3 of yeast . Of all known mobile elements this class shows the closest sequence similarity to retroviruses and has the same arrangement of genes as simpler retroviruses. Hum Mol Genet, 1995 Jan, 4(1), 31 - 6 Molecular dissection of a contiguous gene syndrome: localization of the genes involved in the Langer-Giedion syndrome; Ludecke HJ et al.; The Langer-Giedion syndrome (tricho-rhino-phalangeal syndrome type II, TRPS II) is characterized by craniofacial dysmorphism and skeletal abnormalities . It combines the clinical features of TRPS I and multiple cartilaginous exostoses (EXT) . We have used YAC cloning, Southern blotting, PCR analysis, and fluorescence in situ hybridization to study chromosome 8 deletions, translocations, an inversion, and an insertion in patients with TRPS I, TRPS II or EXT . Our results indicate that the TRPS gene maps more than 1,000 kb proximal to the EXT1 gene and that both genes are affected in TRPS II . We conclude that TRPS II is not due to pleiotropic effects of mutations in a single gene, but that it is a true contiguous gene syndrome. Hum Mol Genet, 1995 Jan, 4(1), 121 - 8 Evidence of a locus for orofacial clefting on human chromosome 6p24 and STS content map of the region; Davies AF et al.; Orofacial clefting is genetically complex, no single gene being responsible for all forms . It can, however, result from a single gene defect either as part of a syndrome (e.g . van der Woude syndrome, Treacher-Collins syndrome, velo-cardio-facial syndrome) or as an isolated phenotypic effect (e.g . X-linked cleft palate; non-syndromic, autosomal dominant orofacial clefting) . Several studies have suggested that chromosome 6p is a candidate region for a locus involved in orofacial clefting . We have used YAC clones from contigs in 6p25-p23 to investigate three unrelated cases of cleft lip and palate coincident with chromosome 6p abnormalities . Case 1 has bilateral cleft lip and palate and a balanced translocation reported as 46,XY,t(6,7)(p23;q36.1) . Case 2 has multiple abnormalities including cleft lip and palate and was reported as 46,XX,del(6)(p23;pter) . Case 3 has bilateral cleft lip and palate and carries a balanced translocation reported as 46,XX,t(6;9)(p23;q22.3) . We have identified two YAC clones, both of which cross the breakpoint in cases 1 and 3 and are deleted in case 2 . These clones map to 6p24.3 and therefore suggest the presence of a locus for orofacial clefting in this region . The HGP22 and AP2 genes, potentially involved in face formation, have been found to flank this region, while F13A maps further telomeric in 6p24.3/25. Genetics, 1995 Jan, 139(1), 203 - 13 Phenotypic and molecular characterization of SerD, a dominant allele of the Drosophila gene Serrate; Thomas U et al.; The Drosophila gene Serrate (Ser) encodes a transmembrane protein with 14 epidermal growth factor--like repeats in its extracellular domain, which is required for the control of cell proliferation and pattern formation during wing development . Flies hetero- or homozygous for the dominant mutation SerD exhibit scalloping of the wing margin due to cell death during pupal stages . SerD is associated with an insertion of the transposable element Tirant in the 3' untranslated region of the gene, resulting in the truncation of the Ser RNA, thereby eliminating putative RNA degradation signals located further downstream . This leads to increased stability of Ser RNA and higher levels of Serrate protein . In wing discs of wild-type third instar larvae, the Serrate protein exhibits a complex expression pattern, including a strong stripe dorsal and a weaker stripe ventral to the prospective wing margin . Wing discs of SerD third instar larvae exhibit additional Serrate protein expression in the edge zone of the future wing margin, where it is normally not detectable . In these cells expression of wing margin specific genes, such as cut and wingless, is repressed . By using the yeast Gal4 system to induce locally restricted ectopic expression of Serrate in the edge zone of the prospective wing margin, we can reproduce all aspects of the SerD wing phenotype, that is, repression of wing margin-specific genes, scalloping of the wing margin and enhancement of the Notch haplo-insufficiency wing phenotype . This suggests that expression of the Serrate protein in the cells of the edge zone of the wing margin, where it is normally absent, interferes with the proper development of the margin. Biotechniques, 1995 Jan, 18(1), 146 - 51 Scintillating microtitration plates as platform for determination of {3H}estradiol binding constants for hER-HBD; Haggblad J et al.; A novel approach to direct determination of ligand binding constants for the human estrogen receptor hormone binding domain was developed . Recombinantly produced human receptor in yeast extracts was attached to scintillating microtitration plates . Radioligand binding to receptors was determined in a multi-detector scintillation counter designed for the microtitration plate format . The method was employed in equilibrium binding experiments, in binding competition tests and in determination of kinetic rate constants . The results obtained show that the methodology is valid in comparison to previously published data regarding hormone binding characteristics of estrogen receptors . Furthermore, the methodology offers several advantages over previous binding assays because the scintillating microtitration plates constitute both the binding reaction vial and the scintillant for the detection of bound radioactivity. Neuroscience, 1995 Jan, 64(2), 277 - 300 The synaptic vesicle and its targets; Volknandt W; Synaptic vesicles play the central role in synaptic transmission . They are regarded as key organelles involved in synaptic functions such as uptake, storage and stimulus-dependent release of neurotransmitter . In the last few years our knowledge concerning the molecular components involved in the functioning of synaptic vesicles has grown impressively . Combined biochemical and molecular genetic approaches characterize many constituents of synaptic vesicles in molecular detail and contribute to an elaborate understanding of the organelle responsible for fast neuronal signalling . By studying synaptic vesicles from the electric organ of electric rays and from the mammalian cerebral cortex several proteins have been characterized as functional carriers of vesicle function, including proteins involved in the molecular cascade of exocytosis . The synaptic vesicle specific proteins, their presumptive function and targets of synaptic vesicle proteins will be discussed . This paper focuses on the small synaptic vesicles responsible for fast neuronal transmission . Comparing synaptic vesicles from the peripheral and central nervous systems strengthens the view of a high conservation in the overall composition of synaptic vesicles with a unique set of proteins attributed to this cellular compartment . Synaptic vesicle proteins belong to gene families encoding multiple isoforms present in subpopulations of neurons . The overall architecture of synaptic vesicle proteins is highly conserved during evolution and homologues of these proteins govern the constitutive secretion in yeast . Neurotoxins from different sources helped to identify target proteins of synaptic vesicles and to elucidate the molecular machinery of docking and fusion . Synaptic vesicle proteins and their markers are useful tools for the understanding of the complex life cycle of synaptic vesicles. Cytogenet Cell Genet, 1995, 69(3-4), 196 - 200 Human cDNA mapping using a high-resolution R-banding technique and fluorescence in situ hybridization; Korenberg JR et al.; High-resolution fluorescence in situ hybridization (FISH) is now an essential element in both human gene mapping and clinical cytogenetics . To facilitate its application, a series of techniques have been developed using FISH to map DNA probes in the size range of 1-1,000 kb directly on R-banded human chromosomes . Distinctive reverse (R) banding is achieved by staining with chromomycin A3 and distamycin A following in situ hybridization . The use of such counterstains enables simultaneous viewing of both the fluorescent R-bands and in situ hybridization signals by either standard photomicroscopy or an automated image-acquisition system . This method is rapid and reproducibly reveals bands at the 350-700 stage . Further, specific methods for chromosome preparation, hybridization, and signal production have been developed and applied in combination with R-banding . These methods are used for precise chromosomal localization of DNA sequences in sizes ranging from that of cDNA (> 1 kb) through bacterial artificial chromosomes (100-150 kb) to yeast artificial chromosomes (> or = 1 Mb) . These techniques provide high-resolution methods for rapid mapping of human genes, expanding the applications of FISH techniques in basic research and clinical analysis. Immunogenetics, 1995, 42(4), 268 - 74 The human factor H-related gene 2 (FHR2): structure and linkage to the coagulation factor XIIIb gene; Skerka C et al.; The human factor H-related gene 2 (FHR2) encodes a serum protein structurally and immunologically related to complement factor H . We describe the isolation and genomic organization of the human FHR2 gene from a yeast artificial chromosome library . The FHR2 gene is organized in five exons and spans about 7 kilobases (kb) of human genomic DNA . A comparison with the corresponding cDNA sequence (clone DDESK59) shows that the analyzed FHR2 gene has a deleted region within exon 4 . A new splice acceptor site created in the truncated exon indicates that the analyzed gene could be translated to a truncated protein . Further, we demonstrate that the genes for FHR2 and beta subunit of coagulation factor XIII are located in the same 165 kb YAC DNA . Thus, the three structurally related genes FXIIIb, FHR2, and factor H are linked on human chromosome 1 in the regulators of complement activation (RCA) gene cluster . The physical linkage of the FHR2 and the factor H genes provides additional evidence for a close relatedness of complement factor H and the factor H-related proteins . The linkage and the almost exclusive organization in short consensus repeat-containing domains indicates a close evolutionary relationship of the FXIIIb, FHR2, and factor H genes. Biochem Cell Biol, 1995 Jan-Feb, 73(1-2), 51 - 8 A Dictyostelium discoideum gene, which is highly related to mo15 from Xenopus, is expressed during growth but not during development; Michaelis C et al.; We have isolated a cDNA from the cellular slime mold Dictyostelium discoideum encoding a protein that is 52% identical to the Xenopus Mo15 kinase and highly related to the equivalent proteins from human (52% identity), rice (52.7% identity), and yeast (47.6% identity) . Mo15 is responsible for the activation of Cdc2 kinase and is itself a member of the large Cdc2-related family of protein kinases . The Dictyostelium protein is more related to the Xenopus Mo15 protein than it is to either the Dictyostelium Cdc2 or Crp proteins . Southern blot analysis of genomic V12-M2 DNA indicated that mo15 is present as a single copy gene that cross hybridizes with cdc2 at low stringency . Northern blot analysis of RNA from different stages of Dictyostelium development showed that mo15 is only expressed during vegetative cell growth. Mycopathologia, 1995, 129(2), 65 - 72 Induction of novel protein synthesis by opsonized Histoplasma capsulatum ingested by murine peritoneal macrophages; Kamei K et al.; It is known that Histoplasma capsulatum can resist the intraphagolysosomal environment and multiply inside macrophages . This resistance can be closely related to its pathogenicity . The mechanism of this resistance has been investigated, but it has not been clarified as yet . To learn about the metabolic condition of the yeast-form of H . capsulatum (isolates G217B and CDC 105) when ingested by macrophages, we investigated protein synthesis by ingested H . capsulatum with {35S}-methionine labeling . Cycloheximide at 5 to 10 micrograms/ml was used to preferentially inhibit macrophage uptake of {35S}-methionine without affecting H . capsulatum uptake . Protein synthesis by H . capsulatum in medium alone served as a positive control . The negative control consisted of macrophages with ingested heat-killed H . capsulatum . Analysis of cytosols with SDS-PAGE and fluorography disclosed that, respectively for G217B and CDC 105, ingested H . capsulatum synthesized 4 and 5 novel proteins, increased the synthesis of 9 and 17 proteins and decreased the synthesis of 9 and 10 constitutive proteins . Ten of these novel or increased proteins were apparently common to both strains . These metabolic changes in ingested H . capsulatum could reflect its adaptation to the intraphagolysosomal environment of macrophages and its ability to multiply there. J Med Vet Mycol, 1995 Jan-Feb, 33(1), 19 - 25 Induction and detection of delayed dermal hypersensitivity in guinea-pig immunized with Blastomyces dermatitidis lysate and filtrate antigens; Abuodeh RO et al.; Guinea-pigs were immunized using yeast phase antigens (lysate and filtrate preparations) from two strains of Blastomyces dermatitidis (T-58 and Le) . Following a sensitization period, the animals were skin tested on days 40 and 216 using T-58 and Le yeast and mycelial phase lysate and filtrate antigen preparations for the detection of delayed dermal hypersensitivity (DTH) . Using the Friedman's analysis of variance by rank test, significant differences were found in the efficacy of the immunogens to induce DTH in the animals when skin tested on both occasions (P < 0.05) . Optimal reactivity was observed in guinea-pigs immunized with Le yeast lysate (mean axes of induration ranging from 12.0 to 18.8 mm and 7.0 to 18.5 mm on day 40 and 216, respectively) and T-58 yeast filtrate (mean axes of induration ranging from 7.5 to 18.0 mm and 8.0 to 17.0 mm on day 40 and 216, respectively) when the immunogens were administered with adjuvant . When the same data was analysed using the Friedman's test with regard to evaluating the efficacy of the skin test antigens to detect DTH, significant differences were found between them (P < 0.05) with optimal results using the Le mycelial filtrate and yeast lysate antigens (mean axes of induration ranging from 7.0 to 16.5 mm and 7.5 to 14.5 mm, respectively) when skin testing was done on day 40 . When skin testing was done on day 216, the T-58 and Le mycelial lysate antigens gave optimal results (mean axes of induration ranging from 9.0 to 18.5 mm and 7.5 to 15.0 mm, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) C R Seances Soc Biol Fil, 1995, 189(1), 7 - 12 {RAS proteins and related proteins}; Tavitian A; Since the mid-eighties, numerous small G-proteins of the ras gene superfamily have been identified and characterized; more than sixty members are distributed into four subfamilies: ras, rho, rab and ran . Although it appears that, structurally, the products of the ras superfamily are related and implicated in various and diverse intracellular mechanisms: signal transduction, cell cycle, differentiation, cellular traffic, etc., their true and specific functions and their scope and extent are still largely unknown for many of them . Approaches to unravel their function include: determination of their intracellular localization, differential expression in various cell and tissues, phenotypic changes induced by the active forms, studies of the interacting molecules . Recent studies combining advanced mammalian cell biology/biochemistry and genetic studies in yeast might help elucidate the specific role exerted by these proteins. J Reprod Fertil Suppl, 1995, 49, 113 - 21 The search for the Booroola (FecB) mutation; Montgomery GW et al.; Sheep derived from the Booroola Merino strain carry an autosomal mutation (FecB) that increases ovulation rate and litter size . One approach to characterize the genetic mutation is to locate the gene using positional cloning . The locus has been mapped to a region between genes for secreted phosphoprotein 1 (SPP1) and epidermal growth factor (EGF) on sheep chromosome 6 . Analysis of possible candidate genes have excluded a number of genes associated with control of reproduction including genes from chromosome 6 . Attempts to define close flanking markers and clone the region of DNA containing the mutation are now in progress . We have cloned additional markers and developed a linkage map showing that the FecB locus maps towards the centromere on chromosome 6 . We have developed a yeast artificial chromosome (YAC) library for the sheep and begun screening the library to identify large DNA clones spanning the FecB region . These will be used to locate the mutation and shed light on how the mutation increases ovulation rate in Booroola sheep. J Gastroenterol Hepatol, 1995 Jan-Feb, 10(1), 51 - 5 Study on the comparative immunogenicity of a recombinant DNA hepatitis B vaccine containing pre-S components of the HBV coat protein with non pre-S containing vaccines; Yap I et al.; A new recombinant hepatitis B vaccine (SCI-B-VAC), derived from Chinese hamster ovary (CHO) cells and consisting of both the major S protein and the minor pre-S1 and pre-S2 proteins of the viral coat were compared with two yeast-derived vaccines containing only S proteins (B-Hepavac II and Engerix-B) for immunogenicity in human volunteers in a randomized controlled study . Two hundred and ninety-five healthy subjects completed the 12 month follow up . There was no difference in the mean age and sex distribution among the three study groups . Seroconversion rates for all the three groups were similar at months 6, 9 and 12 . However, hepatitis B surface antibody (anti-HBs) geometric mean titres (GMT) were significantly higher with 10 micrograms SCI-B-VAC and 20 micrograms Engerix-B than with 10 micrograms B-Hepavac-II at months 6, 9 and 12 . SCI-B-VAC at month 6 also showed a significantly higher anti-HBs GMT than Engerix-B (295 vs 143 miu/mL, P < 0.02). Biochimie, 1995, 77(1-2), 54 - 61 Enzymatic formation of N2,N2-dimethylguanosine in eukaryotic tRNA: importance of the tRNA architecture; Edqvist J et al.; In eukaryotic tRNA, guanosine at position 26 in the junction between the D-stem and the anticodon stem is mostly modified to N2,N2-dimethylguanosine (m2(2)G26) . Here we review the available information on the enzyme catalyzing the formation of this modified nucleoside, the SAM-dependent tRNA (m2(2)G26)-methyltransferase, and our attemps to identify the parameters in tRNA needed for efficient enzymatic dimethylation of guanosine-26 . The required identity elements in yeast tRNA for dimethylation under in vitro conditions by the yeast tRNA(m2(2)G26)-methyltransferase (the TRM1 gene product) are comprised of two G-C base pairs at positions G10-C25 and C11-G24 in the D-stem together with a variable loop of at least five nucleotides . These positive determinants do not seem to act via base specific interactions with the methyltransferase; they instead ensure that G26 is presented to the enzyme in a favorable orientation, within the central 3D-core of the tRNA molecule . The anticodon stem and loop is not involved in such an interaction with the enzyme . In a heterologous in vivo system, consisting of yeast tRNAs microinjected into Xenopus laevis oocytes, the requirements for modification of G26 are less stringent than in the yeast homologous in vitro system . Indeed, G26 in several microinjected tRNAs becomes monomethylated, while in yeast extracts it stays unmethylated, even after extensive incubation . Thus either the X laevis tRNA(m2(2)G26)-methyltransferase has a more relaxed specificity than its yeast homolog, or there exist two distinct G26-methylating activities, one for G26-monomethylation, and one for dimethylation of G26.(ABSTRACT TRUNCATED AT 250 WORDS) Immunogenetics, 1995, 42(5), 386 - 91 Physical mapping of the human and mouse MOG gene at the distal end of the MHC class Ib region; Pham-Dinh D et al.; Myelin/oligodendrocyte glycoprotein (MOG) is expressed specifically in the central nervous system (CNS) by myelinating glial cells, the oligodendrocytes . The external location of MOG on myelin sheaths and its late expression during myelinogenesis argue for a role of MOG in the completion of myelin and maintenance of its integrity . MOG is a target autoantigen in demyelinating diseases, such as experimental autoimmune encephalomyelitis (EAE) in animals and multiple sclerosis (MS) in humans . We previously located the gene encoding MOG to the major histocompatibility complex (MHC), both in human, by cytogenetics, and in mouse, by analysis of recombinants . To refine the position, we have now selected yeast artificial chromosome clones (YAC) which contain the MOG gene . Physical mapping of the human MOG and the mouse Mog genes by characterization of these YAC clones indicated that the gene is located at the distal end of the major histocompatibility complex (MHC) class Ib region in both species . The human MOG gene lies 60 kilobases (kb) telomeric to HLA-F in a head-to-head orientation; the mouse Mog gene lies 25 (kb) telomeric to H2-M5 in a tail-to-head orientation . These orthologous genes provide markers for comparative analysis of the evolution of the MHC in the two species . The physical mapping of MOG should facilitate analysis of its role in hereditary neurological diseases, and the YAC clones identified here will permit the identification of new genes in the region. Cytogenet Cell Genet, 1995, 71(3), 289 - 95 Molecular analysis of a novel subtelomeric repeat with polymorphic chromosomal distribution; Martin-Gallardo A et al.; DNA from a 50-kb yeast artificial chromosome (YAC) containing one human telomere was characterized . Cloned sequences from the centromeric end of this YAC (designated yRM2001) localized to several human chromosomes by somatic hybrid panel mapping . The telomeric end of the YAC contained both (TTAGGG)n sequences and the previously characterized TelBam3.4 subterminal repeat element . A novel low-copy repeat element (designated HC1103) mapped 19 kb from the telomeric end of the YAC . This repeat was shown by fluorescence in situ hybridization to be present in several subtelomeric regions (3q, 12p, 15q, 19p, and 20p) and at an interstitial site (2q13-->q14) in all individuals studied, but to be polymorphically distributed at several other telomeres . The YAC vector-insert EcoRI cloning site was positioned 50 kb to 70 kb from chromosome termini in human genomic DNA using RecA-assisted restriction endonuclease (RARE) cleavage analysis . Our results suggest that the DNA segment cloned in yRM2001 contains a novel block of low-copy DNA consistently present at some human telomeres, but polymorphically distributed at others. Cytogenet Cell Genet, 1995, 71(3), 280 - 4 Mapping the X chromosome breakpoint in two papillary renal cell carcinoma cell lines with a t(X;1)(p11.2;q21.2) and the first report of a female case; Shipley JM et al.; A t(X;1)(p11.2;q21.2) has been reported in cases of papillary renal cell tumors arising in males . In this study two cell lines derived from this tumor type have been used to indicate the breakpoint region on the X chromosome . Both cell lines have the translocation in addition to other rearrangements and one is derived from the first female case to be reported with the t(X;1)(p11.2;q21.2) . Fluorescence in situ hybridization (FISH) has been used to position YACs belonging to contigs in the Xp11.2 region relative to the breakpoint . When considered together with detailed mapping information from the Xp11.2 region the position of the breakpoint in both cell lines was suggested as follows: Xpter-->Xp11.23-OATL1-GATA1-WAS-TFE3-SY P-t(X;1)-DXS255-CLCN5-DXS146-OATL2- Xp11.22-->Xcen . The breakpoint was determined to lie in an uncloned region between SYP and a YAC called FTDM/1 which extends 1 Mb distal to DXS255 . These results are contrary to the conclusion from previous FISH studies that the breakpoint was near the OATL2 locus, but are consistent with, and considerably refine, the position that had been established by molecular analysis. Cytogenet Cell Genet, 1995, 71(3), 268 - 75 A physical map of the region spanning the chromosome 12 translocation breakpoint in a mesothelioma with a t(X;12)(q22;p13); Aerssens J et al.; We have constructed a physical map of a 4.6-cM region of human chromosome band 12p13.3 that contains a translocation breakpoint from a mesothelioma with a t(X;12)(q22;p13) . The map contains a contig of 22 yeast artificial chromosomes (YACs), onto which we have placed 18 sequence tagged site (STS) markers, including seven genes: D12S370, FGF6, KCAN1, KCNA5, KCNA6, NTF3, and VWF . A second YAC contig, comprised of 22 YAC clones, was located distal to the mesothelioma breakpoint and contained 12 STS markers, including four genes (CACNL1A1, D12S380E, D12S381E, and D12S382E) . Based on STS content and fluorescence in situ hybridization experiments, two stable, nonchimeric YAC clones were found that span the mesothelioma breakpoint . A long-range restriction map of an 800-kb region was constructed and used to refine the mesothelioma breakpoint to a region of approximately 100 kb, flanked by the potassium channel genes KCNA1 and KCNA5 . The latter was confirmed by direct visual hybridization (DIRVISH) experiments, using cosmids isolated for markers flanking the breakpoint as probes. Cytogenet Cell Genet, 1995, 71(3), 214 - 6 Physical evidence for the position of the Friedreich's ataxia locus FRDA proximal to D9S5; Hillermann R et al.; Orientation of the Friedreich's ataxia locus (FRDA) with respect to D9S15 and D9S5 has proved critical to the design of subsequent cloning strategies . The rarity of recombination events between FRDA and these markers, originally used to determine assignment to human chromosome region 9q13-->q21.1, has necessitated the instigation of physical mapping studies to determine order and, hence, the precise location of the disease gene . Simultaneous fluorescence in situ hybridisation using cosmid clones located in close proximity to the ends of a 1.2-Mb yeast artificial chromosome clone extending into the FRDA candidate region provides physical evidence for the order of the marker loci to be cen-D9S202-D9S5-D9S15-qter . The possibility that a pericentric inversion, occurring naturally in approximately 1% of the normal population, may affect the order of markers within this region has been eliminated . Considered in association with the interpretation of a recombination event detected in a single affected individual, these data indicate that the FRDA locus is located proximal to D9S5. Eur J Hum Genet, 1995, 3(3), 168 - 79 Expression of the human Dp 71 (apo-dystrophin-1) gene from a 760-kb DMD-YAC transferred to mouse cells; Heikoop JC et al.; A 760-kb YAC was constructed by homologous recombination in yeast, containing the genes located in the distal portion of the DMD gene . The YAC was introduced in mouse LA-9 cells by PEG-mediated cell fusion . One transformant accommodated an intact DMD-YAC, i.e . a full copy of the DMD internal Dp 116, Dp 71 and Dp 40 genes (apo-dystrophin-2, -1 and -3, respectively) . We have studied the expression of the various gene products derived from the introduced DMD-YAC . RT-PCR revealed expression of human Dp 71 but not of Dp 116 or Dp 40 . Remarkably, differences were observed in processing of the 3' region of the endogenous mouse and the human transcripts, due to different splicing of exons 71 (absent in human and present in mouse transcript) and 78 (present in human and absent in mouse transcript) . The splicing pattern of the human transcript is the same as that of the major Dp 71 (apo-dystrophin-1) product in human blood . The observed splicing differences may be caused by either species-specific exon use and/or by cis-acting factors, e.g . the upstream transcript composition, because we have no evidence for endogenous Dp 71 expression. Eur J Cancer, 1995, 31A(4), 527 - 30 Reciprocal translocation at 1p36.2/D1S160 in a neuroblastoma cell line: isolation of a YAC clone at the break; Amler LC et al.; Band 1p36.1-1p36.2 is frequently involved in chromosomal aberrations of neuroblastoma cells, and therefore thought to harbour genetic information which may be involved in tumorigenesis . To map this putative neuroblastoma locus, we screened neuroblastoma cell lines for reciprocal translocations at 1p36.1-2 which may signal the site of an affected gene . We identified a reciprocal 1;15 translocation in cell line NGP by fluorescence in situ hybridisation (FISH) . As a strategy to clone the translocation breakpoint, we isolated yeast artificial chromosomes (YACs) specific for loci at 1p36 . Screening of cell line NGP by FISH identified a YAC, 1050 kbp in size, which hybridised to both derivative 1;15 and 15;1 chromosomes . We conclude that this YAC, which maps to D1S160, covers the break . This chromosomal position is within the smallest region of overlap (SRO) found in neuroblastoma tumours and within the region of a constitutional interstitial deletion of a neuroblastoma patient . The YAC we describe here should serve as a DNA source for gene cloning approaches towards the isolation of candidates for the putative neuroblastoma suppressor gene. Arch Anat Cytol Pathol, 1995, 43(3), 140 - 6 {Colonic Histoplasma capsulatum pseudotumor in AIDS . Morphological and immunohistochemical diagnosis of an isolated lesion}; Hofman P et al.; The authors report a case of a 35-year-old man with acquired immunodeficiency syndrome (AIDS) and a left colonic mass with Histoplasma capsulatum (H . capsulatum) . The look-up performed looking for disseminated infection was negative . In the absence of positive cultures, the diagnosis was determined morphologically based on the presence of yeast observed by light and electron microscopy . The diagnosis was also verified by positive immunofluorescence using specific anti-Histoplasma antibodies . Gastrointestinal histoplasmosis is a frequent complication of AIDS, particularly in some endemic areas of America . Association with a disseminated mycotic infection is then common . Histoplasmosis is less frequently diagnosed in Europe and isolated involvement of the colon is exceptional . When the mycological study is not performed or is negative, only morphological and immunohistochemical methods are able to establish the diagnosis and eliminate other mycotic diseases occurring during AIDS. Annu Rev Biochem, 1995, 64, 315 - 43 Eukaryotic phospholipid biosynthesis; Kent C; The current status of the biochemistry of phospholipid biosynthesis is presented . The review focuses on the identification and characterization of molecular tools such as purified enzymes and cloned genes and cDNAs for those enzymes . The enzymes discussed are those involved in the biosynthesis of the major phospholipid classes, namely, phosphatidate, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, phosphatidylinositol and its phosphorylated derivatives, and cardiolipin . The review centers on the pathways in mammals and yeast . Novel genetic approaches used to delineate pathways and clone cDNAs are discussed . The regulatory roles played by some of the enzymes involved in controlling the biosynthetic pathways are presented. Wiad Parazytol, 1995, 41(2), 131 - 7 {Viruses of parasitic protozoa}; Kasprzak W et al.; The authors present the actual review on several publications concerning the molecular characterizations of the viruses found in parasitic protozoa such as Giardia, Trichomonas, Leishmania and Entamoeba histolytica . All of the RNA viruses observed in parasitic protozoa showed several similarities and did not considerably differ from the viruses found in simple eukaryotic cells; they closely correspond to dsRNA viruses of yeast . The supposition that the protozoan symbionts detected in laboratories transfer to their hosts in natural conditions seemed to be rational, though, there are no evidences that these symbionts are potential pathogens . However, the opinion reiterates that intestinal protozoa (e.g . Entamoeba histolytica) may serve as vectors for HIV or cofactors of HIV infection . The authors point out that irrespective of the potential role of viruses as vectors in the transfection system for parasitic protozoa, the observed viral system constitutes an unusual experimental system to solve the problems of gene expression. Retina, 1995, 15(3), 248 - 52 Choroidal blastomycosis . A report of two cases; Gottlieb JL et al.; PURPOSE: To review the presentation and course of choroidal blastomycosis, a rare chorioretinal mycotic infection, which results from disseminated blastomycosis . METHODS: Two cases of disseminated blastomycosis with ocular infection limited to the choroid are presented . Each patient was diagnosed through biopsy of skin lesions demonstrating the characteristic histologic features and the budding yeast . RESULTS: Systemic evaluation revealed extensive disseminated disease with involvement of the eye, lung, skin, bone and joint, central nervous system, and genitourinary system . Both patients were successfully treated with intravenous amphotericin B with elimination of ocular and systemic disease . CONCLUSION: Although rare, blastomycosis can result in choroidal mycotic infection in immune competent individuals . Tissue biopsy to confirm the diagnosis and extensive systemic evaluation are required. Mol Biol Rep, 1995, 21(1), 49 - 52 Roles of proteasomes in cell growth; Ichihara A et al.; Proteasomes are large, unique protein complexes catalyzing energy- and ubiquitin-dependent proteolysis . Recent studies have revealed that these complexes are involved in two important cellular functions . One is to make antigen fragments for major histo-compatibility complex (MHC) class I-restricted antigen presentation and the other is to regulate the cell cycle by proteolysis . Here we review only the latter function of proteasomes . Proteasomes are widely distributed in eukaryotic cells, but their levels have been shown to be particularly high in various immature cells, such as cancerous, fetal and lymphoblastic cells, and agents including cell differentiation were found to suppress their expression . These conditions also regulate the expression of ubiquitin genes in a similar way, suggesting that proteasomes act ubiquitin-dependently in their 26S form in immature cells . High levels of proteasomes were found immunochemically in the nuclei of rapidly growing cells, indicating that proteasomes are important for eukaryotic cell growth . Indeed, gene disruptions of most subunits of proteasomes in yeast resulted in total suppression of cell growth and cell death . Short-lived regulatory factors of the cell cycle, such as Fos, p53, Mos, and cyclins are degraded by the proteasome-ubiquitin pathway under phosphorylated or dephosphorylated conditions . Ornithine decarboxylase, which is also a short-lived enzyme and is involved in the early phase of cell growth, is quickly degraded by proteasomes with antizyme, but without ubiquitination . Recently, we found that one of the regulatory factors of 26S proteasomes, p31, is a homologue of Nin1p, whose mutation caused inhibition of the cell cycle in yeast . These results indicate that proteasomes play important roles in regulation of the cell cycle in eukaryotes. Mol Biol Rep, 1995, 21(1), 35 - 41 Catalytic components of proteasomes and the regulation of proteinase activity; Rivett AJ et al.; The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells . It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation . Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the alpha and beta subunits of the simpler proteasome isolated from Thermoplasma acidophilum . Proteasomes have a cylindrical structure composed of four rings of seven subunits . The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes . Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites . Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments . It has been assumed from studies with yeast mutants that beta-type subunits play a catalytic role . Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component . Interestingly, mutants in Pre1, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity . These results taken together confirm a direct catalytic role for these beta-type subunits . Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulated in vivo.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Biol Rep, 1995, 21(1), 21 - 6 Molecular biology of proteasomes; Tanaka K; Eukaryotic proteasomes are unusually large proteins with a heterogeneous subunit composition and have been classified into two isoforms with apparently distinct sedimentation coefficients of 20S and 26S . The 20S proteasome is composed of a set of small subunits with molecular masses of 21-32 kDa . The 26S proteasome is a multi-molecular assembly, consisting of a central 20S proteasome and two terminal subsets of multiple subunits of 28-112 kDa attached to the central part in opposite orientations . The primary structures of all the subunits of mammalian and yeast 20S proteasomes have been deduced from the nucleotide sequences of cDNAs or genes isolated by recombinant DNA techniques . These genes constitute a unique multi-gene family encoding homologous polypeptides that have been conserved during evolution . In contrast, little is yet known about the terminal structures of the 26S proteasome, but the cDNA clonings of those of humans are currently in progress . In this review, I summarize available information of the structural features on eukaryotic 20S and 26S proteasomes which has been clarified by molecular-biological methods. Med Trop (Mars), 1995, 55(2), 165 - 71 {Pseudo-parasites in histology and cytopathology}; Pierre C et al.; When interpreting smears and specimens, histologist and cytopathologists can be misled by images mimicking micro-organisms especially parasites such as protozoa, mycotic agents or helminths . Although some of these pitfalls are well-known, others can be problematic especially if nature of the contaminant is the same as that of the parasite that it mimics . False protozoa parasites can correspond either to exogenous agents such as spores, remnants of human cells, or inert exogenous particles . Pseudo-yeast images can be due to pollen, starch or soot but especially to cells such as macrophages, spermatozoids, and neurons or to various inert bodies such as pigments or calcifications . Pseudomycotic filaments can result from vegetable silk, asbestos bodies, radiate granules or fibrin . Curschmann's spirals and vegetable fibers can be confused with helminths and bacterial particles or pollen with helminth eggs. Eur J Hum Genet, 1995, 3(2), 96 - 101 Isolation of microsatellites from the spinal muscular atrophy (SMA) candidate region on chromosome 5q and linkage analysis in Spanish SMA families; Velasco E et al.; A locus responsible for autosomal recessive spinal muscular atrophy (SMA) on chromosome 5q11.2-q13.3 has been mapped to a critical interval delimited by markers D5S435 and D5S557 . By a modification of the Vectorette-(GT)n method, we have isolated three polymorphic CA repeats from two YACs of the SMA region . Two of them (D5S1417 and D5S1416) map within the SMA critical region, and the other (D5S1415) is centromeric to D5S435 . Linkage analysis in Spanish SMA families with eleven markers showed that in our families the disease is linked to this region and confirmed that the novel markers are tightly linked to the SMA locus . The most likely order of markers was 5cen-(D5S63/D5S1356)-(D5S125/D5S465)- (D5S435/D5S1417/D5S1416/D5S557)-D5S610- D5S112-D5S127-5qter, with odds against alternative orders > 1,000:1 . Genetic distances are in agreement with those previously published . However, the recombination fraction between D5S610 and D5S112 is remarkably greater than expected from the physical distance, suggesting a hot spot for recombination in this region . Our results from haplotype and multipoint analyses show that the SMA locus must lie between D5S465 and D5S112, and lend further support to the current location of the SMA locus. Eur J Hum Genet, 1995, 3(2), 87 - 95 A provisional transcript map of the spinal muscular atrophy (SMA) critical region; van der Steege G et al.; YACs from the region containing the spinal muscular atrophy (SMA) locus at 5q12 have been used as probes in a direct screening of cDNA libraries to isolate 8 cDNAs, mapped to different YAC fragments . Three clones showed complete identity to the genes for cyclin B1 (CCNB1), the p44 subunit of the transcription factor BTF2 (BTF2p44), and cofilin (CFL) . Two clones showed partial identity to the beta-glucuronidase gene (GLCB) and a rat integral membrane glycoprotein gene (RNINMEGLA) . CFL turned out to have been identified by a pseudogene sequence . Related sequences occurred on other chromosomes . CCNB1 and BTF2p44 were given an exact location . The GLCB-like gene and the RNINMEGLA-like gene detected loci on both 5q and 5p . The remaining three cDNA clones were localized to the SMA region only . Their sequences did not show identity to any gene for which a function is already known . Two of them have now turned out to be identical to recently reported candidate genes for SMA. Eur J Hum Genet, 1995, 3(2), 78 - 86 Cosmid contigs from the tuberous sclerosis candidate region on chromosome 9q34; van Slegtenhorst M et al.; Tuberous sclerosis (TSC) is a heterogeneous multisystem disorder with loci on 9q34 (TSC1) and 16p13.3 (TSC2) . The TSC2 gene has recently been isolated, while the TSC1 gene has been mapped to a 5-cM region between the markers D9S149 and D9S114 . In our effort to localise and clone TSC1, we have obtained three adjacent cosmid contigs that cover the core of the candidate region . The three contigs comprise approximately 600 kb and include 80 cosmids, 2 P1 clones, 1 YAC, 5 anonymous markers and 4 sequence-tagged sites . The ABO blood group locus, the Surfeit gene cluster, the dopamine beta-hydroxylase gene (DBH) and VAV2, a homologue of the vav oncogene, have all been mapped within the contigs . Exon trapping and mutation screening experiments, aimed at identifying the TSC1 gene, are currently in progress. Biol Cell, 1995, 83(2-3), 105 - 20 Cellular effects of olomoucine, an inhibitor of cyclin-dependent kinases; Abraham RT et al.; Olomoucine (2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine) has been recently described as a competitive inhibitor (ATP-binding site) of the cell cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP-kinase . The unusual specificity of this compound towards cell cycle regulating enzymes suggests that it could inhibit certain steps of the cell cycle . The cellular effects of olomoucine were investigated in a large variety of plant and animal models . This compound inhibits the G1/S transition of unicellular algae (dinoflagellate and diatom) . It blocks Fucus zygote cleavage and development of Laminaria gametophytes . Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine . By arresting cleavage it blocks the Laminaria gametophytes . Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine . By arresting cleavage it blocks the development of Calanus copepod larvae . It reversibly inhibits the early cleavages of Caenorhabditis elegans embryos and those of ascidian embryos . Olomoucine inhibits the serotonin-induced prophase/metaphase transition of clam oocytes; furthermore, it triggers the the release of these oocytes from their meiotic metaphase I arrest, and induces nuclei reformation . Olomoucine slows down the prophase/metaphase transition in cleaving sea urchin embryos, but does not affect the duration of the metaphase/anaphase and anaphase/telophase transitions . It also inhibits the prophase/metaphase transition of starfish oocytes triggered by various agonists . Xenopus oocyte maturation, the in vivo and in vitro phosphorylation of elongation factor EF-1 are inhibited by olomoucine . Mouse oocyte maturation is delayed by this compound, whereas parthenogenetic release from metaphase II arrest is facilitated . Growth of a variety of human cell lines (rhabdomyosarcoma cell lines Rh1, Rh18, Rh28 and Rh30; MCF-7, KB-3-1 and their adriamycin-resistant counterparts; National Cancer Institute 60 human tumor cell lines comprising nine tumor types) is inhibited by olomoucine . Cell cycle parameter analysis of the non-small cell lung cancer cell line MR65 shows that olomoucine affects G1 and S phase transits . Olomoucine inhibits DNA synthesis in interleukin-2-stimulated T lymphocytes (CTLL-2 cells) and triggers a G1 arrest similar to interleukin-2 deprivation . Both cdc2 and cdk2 kinases (immunoprecipitated from nocodazole- and hydroxyurea-treated CTLL-2 cells, respectively) are inhibited by olomoucine . Both yeast and Drosophila embryos were insensitive to olomoucine . Taken together the results of this Noah's Ark approach show that olomoucine arrests cells both at the G1/S and the G2/M boundaries, consistent with the hypothesis of a prevalent effect on the cdk2 and cdc2 kinases, respectively. Biochimie, 1995, 77(1-2), 113 - 24 tRNAs as primer of reverse transcriptases; Marquet R et al.; Genetic elements coding for proteins that present amino acid identity with the conserved motifs of retroviral reverse transcriptases constitute the retroid family . With the exception of reverse transcriptases encoded by mitochondrial plasmids of Neurospora, all reverse transcriptases have an absolute requirement for a primer to initiate DNA synthesis . In retroviruses, plant pararetroviruses, and retrotransposons (transposons containing long terminal repeats), DNA synthesis is primed by specific tRNAs . All these retroelements contain a primer binding site presenting a Watson-Crick complementarity with the primer tRNA . The tRNAs most widely used as primers are tRNA(Trp), tRNA(Pro), tRNA(1,2Lys), tRNA(3Lys), tRNA(iMet) . Other tRNAs such as tRNA(Gln), tRNA(Leu), tRNA(Ser), tRNA(Asn) and tRNA(Arg) are also occasionally used as primers . In the retroviruses and plant pararetroviruses, the primer binding site is complementary to the 3' end of the primer tRNA . In the case of retrotransposons, the primer binding site is either complementary to the 3' end or to an internal region of the primer tRNA . Additional interactions taking place between the primer tRNA and the retro-RNA outside of the primer binding site have been evidenced in the case of Rous sarcoma virus, human immunodeficiency virus type I, and yeast retrotransposon Ty1 . A selective encapsidation of the primer tRNA, probably promoted by interactions with reverse transcriptase, occurs during the formation of virus or virus-like particles . Annealing of the primer tRNA to the primer binding site appears to be mediated by reverse transcriptase and/or the nucleocapsid protein . Modified nucleosides of the primer tRNA have been shown to be important for replication of the primer binding site, encapsidation of the primer (in the case of Rous sarcoma virus), and interaction with the genomic RNA (in the case of human immunodeficiency virus type I). Eur J Hum Genet, 1995, 3(1), 14 - 20 Deletion and insertion mutations in short tandem repeats in the coding regions of human genes; Darvasi A et al.; In vitro studies in bacterial, yeast and eukaryotic systems have demonstrated the existence of deletion and insertion 'hot-spots' involving repetitive sequences . Slipped-strand mispairing (SSM) has been suggested to be the mechanism involved . Progress in human molecular genetics has allowed the identification of many mutations causing diseases . Analysis of sequences involved in these mutations provides an opportunity to investigate the contribution of short tandem repeats to the naturally occurring mutations in coding regions of human genes . We have analyzed the sequences surrounding 625 disease-causing mutations in the coding regions of three genes: the cystic fibrosis transmembrane conductance regulator, beta globin and factor IX . Altogether, 134 (21%) insertion and deletion mutations of 4 base pairs or less were identified . In 47% of these mutations, the deletions and insertions occurred within a unit repeated tandemly 2- to 7-fold . These were classified as SSM mutations . The proportion of SSM mutations was significantly higher than expected by chance . The estimated net proportion of deletion and insertion mutations attributed to SSM was 27% . These results indicate that very short repetitive sequences contribute significantly to the generation of deletion and insertion mutations in human genes, and to the evolution of diversity of their coding regions. J Pharm Biomed Anal, 1995 Jan, 13(1), 9 - 20 In vitro bioassay with enhanced sensitivity for human granulocyte colony-stimulating factor; Hammerling U et al.; A method for the determination of human granulocyte colony-stimulating factor (hG-CSF) activity, based on stimulation of cellular proliferation, was developed using a subclone of the murine myeloid leukemia cell line NFS-60, with an improved sensitivity for hG-CSF, as indicator . The optimal range for quantitative analysis of hG-CSF was about 4-60 pg ml-1 . The stimulatory effect was measured by a colorimetric microassay: the optical density of formazan, which is produced by viable cells from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), was obtained by reading plates in a multi-channel photometer . The assay was designed as a five-dose parallel line test, employing three or four doses for potency determinations, which fulfil pharmacopoeial requirements for assay validity . Inter-assay relative standard deviation (RSD) varied between 5.2 and 12.0% . Most assay experiments revealed potencies within limits of error of 90-110% and the mean index of precision value was 0.057 . The recently developed yeast cell-derived International Standard (88/502) served as a reference for activity of rhG-CSF . Specificity of the assay was demonstrated by absence of response upon exposure to a panel of biomolecules, including recombinant human interleukin-3, and by the suppression of growth stimulation in the presence of neutralizing anti hG-CSF antibodies . Potency readings of unglycosylated rhG-CSF were dependent on pH of assay medium with higher relative activities observed at pH 6.6 than at 7.4 . Moreover, SDS-PAGE analysis of the carbohydrate-deficient preparation, following incubation at physiological pH, revealed several high molecular weight rhG-CSF bands and decreased monomeric form . The method described was found suitable for potency assessments of pharmaceutical formulations of hG-CSF. Mutat Res, 1995 Jan, 326(1), 39 - 49 Physical mapping of the human hprt chromosomal region (Xq26); Lippert MJ et al.; The human hprt chromosomal region (Xq26) was physical-mapped using pulsed field gel electrophoresis (PFGE) . This work involved: (i) the recovery of three new genomic DNA markers (DXS1327, DXS1328, and DXS1329), (ii) the ordering of new markers relative to 11 previously available hprt-linked markers by deletion mapping, and (iii) the completion of human T-lymphocyte PFGE Southern blots using the 14 Xq26 markers . A contiguous 1.5-Mb physical map of the region telomeric to hprt was determined . As this map identifies clusters of in vivo unmethylated rare-cutter restriction sites, potential CpG islands are revealed. Mol Cell Biol, 1995 Jan, 15(1), 326 - 37 Activation of pp70/85 S6 kinases in interleukin-2-responsive lymphoid cells is mediated by phosphatidylinositol 3-kinase and inhibited by cyclic AMP; Monfar M et al.; Activation of phosphatidylinositol 3-kinase (PI3K) and activation of the 70/85-kDa S6 protein kinases (alpha II and alpha I isoforms, referred to collectively as pp70S6k) have been independently linked to the regulation of cell proliferation . We demonstrate that these kinases lie on the same signalling pathway and that PI3K mediates the activation of pp70 by the cytokine interleukin-2 (IL-2) . We also show that the activation of pp70S6k can be blocked at different points along the signalling pathway by using specific inhibitors of T-cell proliferation . Inhibition of PI3K activity with structurally unrelated but highly specific PI3K inhibitors (wortmannin or LY294002) results in inhibition of IL-2-dependent but not phorbol ester (conventional protein kinase C {cPKC})-dependent pp70S6k activation . The T-cell immunosuppressant rapamycin potently antagonizes IL-2-(PI3K)- and phorbol ester (cPKC)-mediated activation of pp70S6k . Thus, wortmannin and rapamycin antagonize IL-2-mediated activation of pp70S6k at distinct points along the PI3K-regulated signalling pathway, or rapamycin antagonizes another pathway required for pp70S6k activity . Agents that raise the concentration of intracellular cyclic AMP (cAMP) and activate cAMP-dependent protein kinase (PKA) also inhibit IL-2-dependent activation of pp70S6k . In this case, inhibition appears to occur at least two points in this signalling path . Like rapamycin, PKA appears to act downstream of cPKC-mediated pp70S6k activation, and like wortmannin, PKA antagonizes IL-2-dependent activation of PI3K . The results with rapamycin and wortmannin are of added interest since the yeast and mammalian rapamycin targets resemble PI3K in the catalytic domain. Vaccine, 1995, 13(8), 699 - 702 Molecular epidemiology of hepatitis B virus vaccine variants in Singapore; Oon CJ et al.; Perinatal infection with variants of hepatitis B virus occurs despite combined immunoprophylaxis with hepatitis B immunoglobulin and currently licensed plasma-derived and recombinant yeast hepatitis B vaccines . Several variants have been detected during a large study of infants born to carrier mothers in Singapore . The most frequent variant was a virus in which a single amino acid substitution Gly to Arg occurred at amino acid position 145 of the outer protein coat of the virus . Similar mutations have been described in Italy, Japan, the USA and a number of other countries . The emergence of antibody escape mutants is a cause for concern for the detection of virus and possibly for future immunization programmes. Virus Genes, 1995, 10(1), 99 - 102 HBx protein of hepatitis B virus interacts with the C-terminal portion of a novel human proteasome alpha-subunit; Fischer M et al.; Two-hybrid protein interaction screening in yeast was used to identify proteins that interact with the HBx nonstructural protein of hepatitis B virus (HBV) . A new human member of the proteasome alpha-subunit family was obtained . Its protein sequence closely resembles the 28 kD subunits from other organisms . The interaction with HBx was abolished by a two amino-acid insertion behind position 128 in HBx, in a region previously found to be essential for its transcriptional transactivation function . These data support a model of HBx acting indirectly on transcriptional processes . By binding to a specific proteasome alpha-subunit, HBx might interfere with degradative processes, thereby enhancing the half-life of different transcription factors and other nuclear regulatory proteins . Interaction with the Hu 28k proteasome subunit could thus provide a unifying explanation for the markedly pleiotropic effects of HBx. Gene, 1994 Dec 30, 151(1-2), 309 - 14 Structural organisation and chromosomal mapping of the human Id-3 gene; Deed RW et al.; The helix-loop-helix (HLH) family of transcription factors plays a central role in the regulation of cell growth, differentiation and tumourigenesis . Members of the Id (inhibitor of DNA binding) class of these nuclear proteins are able to heterodimerise with and thereby antagonise the functions of other transcription factors of this family . We report here on the genomic organisation of the human Id3 (HLH 1R21/heir1) gene . Comparison with the two other mammalian Id genes, Id1 and Id2, reveals a highly conserved protein coding gene organisation consistent with evolution from a common, ancestral Id-like gene . In addition, by using a yeast artificial chromosome (YAC) clone of Id3, we have fine-scale mapped the gene to chromosome band 1p36.1 by fluorescence in situ hybridisation (FISH) and, using the same FISH technique, we have detected heterogeneity in tumour-associated 1p36 chromosome translocations. J Biol Chem, 1994 Dec 30, 269(52), 32759 - 67 The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06-A resolution; Sundaramoorthy M et al.; The crystal structure of manganese peroxidase (MnP) from the lignin-degrading basidiomycetous fungus Phanerochaete chrysosporium has been solved using molecular replacement techniques and refined to R = 0.20 at 2.0 A . The overall structure is similar to that of two other fungal peroxidases, lignin peroxidase from P . chrysosporium and Arthromyces ramosus peroxidase . Like the other fungal peroxidases, MnP has two structural calcium ions . MnP also has two N-acetylglucosamine residues N-linked to Asn131 that are readily visible in the electron density map . The active site, consisting of a proximal His ligand H-bonded to an Asp residue and a distal side peroxide binding pocket consisting of a catalytic His and Arg, is the same as in the aforementioned fungal peroxidases as well as yeast cytochrome c peroxidase . MnP differs in having five rather than four disulfide bonds . The additional disulfide bond, Cys341-Cys348, is located near the C terminus of the polypeptide chain . Importantly, a new cation binding site, which we propose is the manganese-binding site of MnP, was located in the crystal structure . The ligands constituting the Mn(2+)-binding site include Asp179, Glu35, Glu39, a heme propionate, and two water molecules . Electron transfer from Mn2+ to the heme edge or iron center is envisioned to occur through a sigma-bonded pathway along a heme propionate. Biochim Biophys Acta, 1994 Dec 30, 1188(3), 367 - 72 Primary structure, cell-free synthesis and mitochondrial targeting of the 8.2 kDa protein of cytochrome c reductase from potato; Braun HP et al.; Cytochrome c reductase from potato comprises ten subunits with apparent molecular sizes between 55 and < 10 kDa . The subunit with the highest electrophoretic mobility on SDS-polyacrylamide gels was isolated and analysed by cyclic Edman degradation . Mixtures of degenerative oligonucleotides were derived from the obtained sequence data and used for the isolation of corresponding cDNA clones . The clones encode a protein of 72 amino acids which exhibits significant sequence identity with a 9.5 kDa subunit of cytochrome c reductase from bovine and a 11 kDa subunit of the enzyme complex from yeast . Comparison between the deduced amino acid sequence of the open reading frame and the sequence of the mature protein reveals that only the initiator methionine is absent in the functional subunit . Hence the protein has a calculated molecular mass of 8.2 kDa . Transcripts of the potato 8.2 kDa protein were not translated in reticulocyte lysates but in vitro translation worked efficiently with wheat germ lysate . Import of the radiolabelled protein into isolated mitochondria from potato seems to depend on a potential across the inner membrane and confirms the absence of a cleavable mitochondrial presequence. J Biol Chem, 1994 Dec 23, 269(51), 32144 - 50 Analysis of an invariant cofactor-protein interaction in thiamin diphosphate-dependent enzymes by site-directed mutagenesis . Glutamic acid 418 in transketolase is essential for catalysis; Wikner C et al.; A homologous expression system and a purification protocol for pure, highly active recombinant yeast transketolase have been developed . The invariant transketolase residue Glu418, which forms a hydrogen bond to the N-1' nitrogen atom of the pyrimidine ring of the cofactor thiamin diphosphate has been replaced by glutamine and alanine . Crystallographic analyses of the mutants show that these amino acid substitutions do not induce structural changes beyond the site of mutation . In both cases, the cofactor binds in a manner identical to the wild-type enzyme . Significant differences in the CD spectra of the mutant transketolases compared with the spectrum of wild-type enzyme indicate differences in the electron distribution of the aminopyrimidine ring of the cofactor . The E418Q mutant shows 2% and the E418A mutant shows about 0.1% of the catalytic activity of wild-type enzyme . The affinities of the mutant enzymes for thiamin diphosphate are comparable with wild-type transketolase . The hydrogen bond between the coenzyme and the side chain of Glu418 is thus not required for coenzyme binding but essential for catalytic activity . The results demonstrate the functional importance of this interaction and support the molecular model for cofactor deprotonation, the first step in enzymatic thiamin catalysis. Nature, 1994 Dec 22-29, 372(6508), 798 - 800 Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1; Yan M et al.; A kinase distinct from the MEK activator Raf, termed MEK kinase-1 (MEKK), was originally identified by virtue of its homology to kinases involved in yeast mating signal cascades . Like Raf, MEKK is capable of activating MEK in vitro . High-level expression of MEKK in COS-7 cells or using vaccinia virus vectors also activates MEK and MAPK, indicating that MEKK and Raf provide alternative means of activating the MAPK signalling pathway . We have derived NIH3T3 cell sublines that can be induced to express active MEKK . Here we show that induction of MEKK does not result in the activation of MAPK, but instead stimulates the stress-activated protein kinases (SAPKs) which are identical to a Jun amino-terminal kinase . We find that MEKK regulates a new signalling cascade by phosphorylating an SAPK activator, SEK1 which in turn phosphorylates and activates SAPK. Proc Natl Acad Sci U S A, 1994 Dec 20, 91(26), 12629 - 33 Binding of Vav to Grb2 through dimerization of Src homology 3 domains; Ye ZS et al.; The protooncogenic protein Vav has the structure of an intracellular signal transducer . It is exclusively expressed in cells of hematopoietic lineage and plays a crucial role in hematopoietic cell differentiation . Here we report that both in cell extracts and within intact mammalian cells Vav binds to Grb2 (Sem-5/ASH/Drk), an adaptor molecule which plays a key role in Ras activation . The interaction became evident from a yeast two-hybrid screen and its specificity was demonstrated by in vitro binding assays . It is mediated by an unusual protein-protein binding reaction: dimerization of specific intact Src homology 3 domains of each of the partners . Signaling during hematopoietic lineage differentiation may therefore involve the tissue-specific signal transducer Vav linking into the ubiquitous pathway involving Grb2 and ultimately Ras. Proc Natl Acad Sci U S A, 1994 Dec 20, 91(26), 12609 - 13 Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras, H-ras, K-ras, and Rap; Spaargaren M et al.; To identify proteins that bind to the Ras-related protein R-ras we performed a yeast two-hybrid cDNA library screen . Several clones were obtained encoding the C-terminal region of the guanine nucleotide dissociation stimulator for Ral (RalGDS) . The R-ras-binding domain of RalGDS (RalGDS-RBD) is distinct from the conserved catalytic exchange factor regions . Using the two-hybrid system, we show that RalGDS-RBD interacts with H-ras, K-ras, and Rap, and with active but not with inactive point mutants of these Ras-like GTPases . Moreover, using purified proteins, we demonstrate the direct GTP-dependent interaction of the Ras-like GTPases with RalGDS-RBD and full-length RalGDS in vitro . Furthermore, we show that RalGDS-RBD and the Ras-binding domain of Raf-1 compete for binding to the Ras-like GTPases . These data indicate that RalGDS is a putative effector molecule for R-ras, H-ras, K-ras, and Rap. J Biol Chem, 1994 Dec 16, 269(50), 31777 - 84 Molecular characterization of the 77-kDa echinoderm microtubule-associated protein . Homology to the beta-transducin family; Li Q et al.; The major microtubule-associated protein (MAP) of sea urchins and several other echinoderms is a polypeptide of M(r) 77,000 . The echinoderm MAP (EMAP) is abundant in embryonic and differentiated cells, as well as in mitotic and interphase microtubule arrays . To characterize the molecular structure and function of the EMAP, we isolated a full-length cDNA clone, which has one open reading frame that predicts a polypeptide of 686 amino acids with a calculated M(r) of 75,488 . On the basis of charge distribution, EMAP can be divided into two distinctive domains: The NH2-terminal basic region (amino acids 1-137, pI = 10.0) and a slightly acidic, COOH-terminal region (amino acids 138-686, pI = 5.8) . This charge distribution is typical of many microtubule-binding proteins, but no significant sequence homology has been detected with any known MAPs . The EMAP, however, does show significant sequence similarity with the beta-subunit of the heterotrimeric G-protein, transducin . The homology lies in a series of 10 imperfect, 43-amino acid repeats (WD-40 repeats) that have been found in many proteins of diverse functions, including beta-transducins, Drosophila Enhancer of split, the yeast STE4, CDC4, CDC20, PRP4, and Tup1 gene products, and the dTAFII80 subunit of Drosophila TFIID . The function of these repeats still remains unknown . It is possible that these repeats are involved in protein-protein interactions, perhaps with the tetratricopeptide repeat-containing protein family . Alternatively, the EMAP may be an important link between signal transduction events and a change in microtubule organization during the cell cycle. J Biol Chem, 1994 Dec 16, 269(50), 31563 - 6 Role of hydrophobicity of phenylalanine beta 85 and leucine beta 88 in the acceptor pocket for valine beta 6 during hemoglobin S polymerization; Adachi K et al.; Characterization of the hydrophobic EF acceptor pocket involving Phe-beta 85 and Leu-beta 88 as well as the Val-beta 6 donor site is critical for understanding the polymerization of deoxy Hb S . Glu substitutions at beta 85 or beta 88 in Hb S were made and expressed in yeast in an effort to evaluate the role of hydrophobicity in the acceptor pocket during polymerization of Hb S . Both substitutions result in decreased tetramer stability, increases in oxygen affinity, and inhibition in polymerization compared with Hb S . Critical concentrations for polymerization of Hb SF beta 85E and Hb SL beta 88E were 2.4- and 7-fold higher, respectively, than that of Hb S, while the value for Hb SL beta 88E was intermediate between those previously reported for Hb SL beta 88A and Hb SL beta 88F (Adachi, K., Konitzer, P., Paulraj, C . G., and Surrey, S . (1994) J . Biol . Chem . 269, 17477-17480) . Kinetics of polymerization of Glu-beta 85 and Glu-beta 88 deoxy Hb S tetramers were biphasic at lower hemoglobin concentrations like deoxy Hb SL beta 88A, suggesting formation of two types of polymers during polymerization . The time required to form half the total amount of polymer (t1/2) for deoxy Hb SF beta 85E was 10-fold shorter than that for deoxy Hb SL beta 88E . In addition, t1/2 for deoxy Hb SF beta 85E was 2.5-fold shorter, while that for Hb SL beta 88E was 4-fold longer than deoxy Hb SL beta 88A at equivalent concentrations . These results suggest that hydrophobicity of the amino acid at beta 88 appears more critical than that at beta 85 in the acceptor pocket for Val-beta 6 . Furthermore, stereospecificity of the acceptor pocket in addition to hydrophobicity of beta 88 are critical for stable hydrophobic interactions with Val-beta 6 during deoxy Hb S polymerization. J Biol Chem, 1994 Dec 16, 269(50), 31518 - 24 Potent transactivation domains of the Ah receptor and the Ah receptor nuclear translocator map to their carboxyl termini; Jain S et al.; The Ah receptor (AHR) is a ligand-activated transcription factor that is structurally related to its dimerization partner, the Ah receptor nuclear translocator (ARNT), and two Drosophila proteins, SIM and PER . All four proteins contain a region of homology now referred to as a PAS homology domain . In addition, the AHR, ARNT, and SIM harbor a basic region helix-loop-helix motif in their N termini, whereas PER does not . Previous mapping studies of the AHR have demonstrated that the PAS domain contains sequences required for ligand recognition, dimerization, and interaction with the 90-kDa heat shock protein . They also have confirmed that the basic region helix-loop-helix domain plays a role in both dimerization and sequence-specific DNA binding . To identify domains involved in transactivation of target genes, we generated chimeras of AHR/ARNT deletion mutants with the DNA binding region of the yeast Gal4 protein, transiently expressed these in COS-1 cells, and monitored their capacity to activate the chloramphenicol acetyltransferase reporter gene under the control of a minimal promoter driven by enhancer elements recognized by Gal4 . Extensive analysis of these fusions revealed that the AHR and ARNT harbor potent transactivation domains within their C termini . Importantly, the amino-terminal halves of both the AHR and ARNT were found to be devoid of transactivation activity. J Biol Chem, 1994 Dec 16, 269(50), 31397 - 403 Coenzyme A dependence of glycosylphosphatidylinositol biosynthesis in a mammalian cell-free system; Stevens VL et al.; The biosynthesis of glycosylphosphatidylinositol (GPI) in mammals and yeast involves a step not observed in trypanosomes . This reaction, which is the inositol acylation of glucosamine phosphatidylinositol (GlcN-PI), occurs as the third step in the biosynthetic pathway . In this study, conditions were developed to stimulate this reaction in vitro . The synthesis of the GlcN-PI(acyl) from either UDP-{6-3H}GlcNAc or {6-3H} GlcNAc-PI by murine lymphoma cell microsomes was greatly enhanced by the addition of either CoA or palmitoyl-CoA . Stimulation of this reaction was optimal with 1 microM of either compound and required that the precursor, GlcN-PI, be synthesized in the presence of GTP, a specific effector of the formation of this glycolipid . That GlcN-PI(acyl) was generated from GlcN-PI was established by pulse-chase analysis . Because no acyl-chain specificity for acyl-CoA stimulation of GlcN-PI(acyl) synthesis was found and attempts to demonstrate direct transfer of {3H}palmitate from {3H}palmitoyl-CoA to the third intermediate in GPI biosynthesis were unsuccessful, the possibility that free CoA was the activator of this reaction was considered . CoA-stimulated GlcN-PI acylation occurred in the absence of ATP, an essential cofactor for acyl-CoA synthesis, indicating that free CoA is the endogenous effector of the third step in mammalian GPI biosynthesis . This finding is consistent with this inositol acylation being catalyzed by a CoA-dependent transacylase . Mannose-containing GPI intermediates were synthesized in vitro when GDP-mannose was added in the presence of GTP and CoA . Therefore, when effectors of the initial reactions in GPI biosynthesis are included, later steps in this pathway can be studied in mammalian cell-free systems. Cancer Res, 1994 Dec 15, 54(24), 6374 - 82 The search for BRCA1; Friedman LS et al.; BRCA1, a gene predisposing to breast and ovarian cancer, was mapped to chromosome 17q21 by linkage analysis . Loss of heterozygosity in breast and ovarian tumors from BRCA1-linked patients always involved loss of wild-type alleles from chromosome 17q21, suggesting that BRCA1 acts as a tumor suppressor gene . Meiotic recombination in linked families constrained the BRCA1 region to an estimated physical size of 650 kilobases . Twenty-two candidate genes were isolated by screening complementary DNA libraries with yeast artificial chromosomes and cosmids from the critical region . Of these, 8 were known human genes, 7 were homologues of genes identified in other species, and 7 encoded novel transcripts . Each gene were sequenced and analyzed for variation, revealing 44 variants, including two missense mutations in two genes which segregated with breast cancer and were not found in controls . However, no frame-shift, nonsense, or regulatory mutations were found. Mol Gen Genet, 1994 Dec 15, 245(6), 760 - 8 Apparent functional independence of the mitochondrial and nuclear transcription systems in cultured human cells; Sewards R et al.; We have constructed a series of reporter constructs which test the effects of sequence elements from the control region of human mitochondrial DNA on expression in the nucleus, as assayed by transient expression in cultured human cells . The mitochondrial heavy-strand promoter (HSP) was unable to function as a promoter in nuclear DNA . Neither the HSP, nor the binding region for the mitochondrial transcription factor mtTF1 from the light-strand promoter, had any significant or systematic modulatory effects upon transcription from strong or weak RNA polymerase II (pol II) promoters, in three different human cell lines . The same finding held true regardless of orientation with respect to the start site of transcription . Similar results were obtained with a rho 0 derivative of one of these lines, indicating that mitochondrial promoter sequences in the nucleus cannot modulate transcription in response to altered mtDNA copy number . These results support the view that the nuclear and mitochondrial transcription systems in human cells are functionally independent, and do not communicate through factors recognizing shared sequence elements, as suggested by studies in yeast. Eur J Biochem, 1994 Dec 15, 226(3), 1007 - 17 Purification and properties of phosphofructokinase from Dictyostelium discoideum; Martinez-Costa OH et al.; Phosphofructokinase (PFruK) from the slime mold Dictyostelium discoideum has been purified to homogeneity over 15,000-fold with a 29% yield . Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the final preparation revealed a single band of 95 kDa . The native molecular mass was determined by gel filtration to be 382 kDa, indicating that the enzyme is a homotetramer . An antibody raised in rabbits against the 95-kDa band immunoprecipitated PFruK activity while it did not react with the enzyme from yeast and mammalian cells . The apparent pI was 6.8 and the pH optimum was 7.6 . The enzyme had an activation energy (Ea) of 29.1 kJ/mol . The amino acid composition was distinctive in having high Ser, Gly and Glx and low Ala, Val and Tyr compared with other eukaryotic PFruKs . Enzyme activity did not have a sigmoidal saturation curve for fructose 6-phosphate, was only mildly inhibited by MgATP at acidic pH values, was not affected by enzyme concentration and was insensitive to any of the typical allosteric effectors of PFruKs from other sources . However, the enzyme binds fructose 2,6-bisphosphate as indicated by protection against thermal denaturation . Treatment with cAMP-dependent protein kinase led to phosphorylation of the enzyme without change in activity . The metabolic significance of these properties and their relationship to structure/function are discussed. EMBO J, 1994 Dec 15, 13(24), 5944 - 57 Immunophilins interact with calcineurin in the absence of exogenous immunosuppressive ligands; Cardenas ME et al.; The peptidyl-prolyl isomerases FKBP12 and cyclophilin A (immunophilins) form complexes with the immunosuppressants FK506 and cyclosporin A that inhibit the phosphatase calcineurin . With the yeast two hybrid system, we detect complexes between FKBP12 and the calcineurin A catalytic subunit in both the presence and absence of FK506 . Mutations in FKBP12 surface residues or the absence of the calcineurin B regulatory subunit perturb the FK506-dependent, but not the ligand-independent, FKBP12-calcineurin complex . By affinity chromatography, both FKBP12 and cyclophilin A bind calcineurin A in the absence of ligand, and FK506 and cyclosporin A respectively potentiate these interactions . Both in vivo and in vitro, the peptidyl-prolyl isomerase active sites are dispensable for ligand-independent immunophilin-calcineurin complexes . Lastly, by genetic analyses we demonstrate that FKBP12 modulates calcineurin functions in vivo . These findings reveal that immunophilins interact with calcineurin in the absence of exogenous ligands and suggest that immunosuppressants may take advantage of the inherent ability of immunophilins to interact with calcineurin. Blood, 1994 Dec 15, 84(12), 4344 - 53 alpha-Amino butyric acid cannot reactivate the silenced gamma gene of the beta locus YAC transgenic mouse; Pace B et al.; Butyric acid, a naturally occurring fatty acid, has been shown to increase fetal hemoglobin in BFUe cultures, in primates, and in patients with beta chain hemoglobinopathies . The precise mechanism of gamma gene induction by butyrate is unknown . Butyrate may induce fetal hemoglobin production in vivo by reactivation of silenced gamma globin genes, by inhibiting the silencing of gamma genes, or by both mechanisms . We examined the effects of butyrate on gamma gene expression in transgenic mice carrying three types of constructs: microLCRA gamma mice, which continue to express the gamma gene in the adult stage of development at a level of one-third to one-fifth of the expression in the fetus; microLCRA gamma psi beta delta beta mice, which display correct developmental regulation of gamma and beta human globin genes and have low level gamma globin expression in the adult; and beta locus YAC mice, which display correct developmental regulation of epsilon, gamma, and beta globin genes and have a totally silenced gamma gene in the adult stage . Animals were treated with a continuous infusion of alpha-amino butyric acid (alpha-ABA) for 7 days . In microLCRA gamma mice alpha-ABA produced up to a 43-fold induction of gamma and 9-fold induction of mouse alpha globin genes . In contrast, butyrate did not induce gamma globin expression in the beta locus YAC mice . However, the gamma globin genes of beta locus YAC mice were activated after administration of 5-azacytidine (5-azaC), and the level of gamma globin expression was further increased by administration of alpha-ABA . These results suggest that butyrate cannot reactivate a totally silenced gamma gene and that induction of fetal hemoglobin by this compound may require the presence of preactivated gamma globin genes. Nucleic Acids Res, 1994 Dec 11, 22(24), 5196 - 203 Molecular charac