Nucleic Acids Res, 1993 Jun 25, 21(12), 2837 - 44 Estimation of protein coding density in a corpus of DNA sequence data; Fickett JW et al.; A number of experimental methods have been reported for estimating the number of genes in a genome, or the closely related coding density of a genome, defined as the fraction of base pairs in codons . Recently, DNA sequence data representative of the genome as a whole have become available for several organisms, making the problem of estimating coding density amenable to sequence analytic methods . Estimates of coding density for a single genome vary widely, so that methods with characterized error bounds have become increasingly desirable . We present a method to estimate the protein coding density in a corpus of DNA sequence data, in which a 'coding statistic' is calculated for a large number of windows of the sequence under study, and the distribution of the statistic is decomposed into two normal distributions, assumed to be the distributions of the coding statistic in the coding and noncoding fractions of the sequence windows . The accuracy of the method is evaluated using known data and application is made to the yeast chromosome III sequence and to C . elegans cosmid sequences . It can also be applied to fragmentary data, for example a collection of short sequences determined in the course of STS mapping. Biochim Biophys Acta, 1993 Jun 24, 1164(1), 17 - 21 Phosphonate inhibitors of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase; Li YK et al.; Several bisphosphonates were examined as inhibitors of yeast GPD (glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.12) and PGK (phosphoglycerate kinase, EC 2.7.2.3) . The phosphonomethyl analog of 2-deoxy-1,3-bisphosphoglycerate (i.e., 2-oxo-1,5-bisphosphonopentane, 2-oxo-PC5P) is a good inhibitor of PGK (Ki = 0.2 +/- 0.08 mM at pH 8.5, 27 degrees C) and a poor inhibitor of GPD (Ki = 20 +/- 1 mM, pH 8.5) . The shorter, butane, analog (2-oxo-PC4P) binds more tightly to PGK (Ki = 84 +/- 6 microM), and about equally well to GPD, as does 2-oxo-PC5P . The 2-oxo-bisphosphonates bind to PGK more tightly (by approx . 4 kJ/mol) than do the corresponding non-carbonyl analogues (1,4-bisphosphonobutane and 1,5-bisphosphonopentane). Brain Res, 1993 Jun 18, 614(1-2), 131 - 6 p34cdc2 homologue is located in nucleoli of the nervous and endocrine systems; Ino H et al.; p34cdc2 protein kinase is a component of M phase-promoting factor (MPF), which plays an important role in controlling the mitotic and meiotic cell cycle . p34cdc2 contains a unique 16 amino acid sequence (PSTAIR) that is conserved from fission yeast to human . Using polyclonal anti-PSTAIR antibody, we detected the p34cdc2 homologue in the central nervous system of adult mice by western blotting . By immunohistochemical technique, we found that the p34cdc2 homologue was located in the nucleoli of neurons and glia in the central and peripheral nervous systems . In the central nervous system, positive cells were widely distributed from the cerebral cortex to the spinal cord . Immunoreactive cells were also detected in retina and pituitary . The evidence that the p34cdc2 is present in neurons which have lost the ability of cell division predicts another function of p34cdc2 family proteins besides the one that has generally recognized. Nature, 1993 Jun 17, 363(6430), 637 - 40 A conserved mitotic kinase active at late anaphase-telophase in syncytial Drosophila embryos; Fenton B et al.; Mutations in the Drosophila gene polo cause abnormal mitotic and meiotic divisions . This gene encodes a 577-amino-acid protein that has an N-terminal putative kinase domain and a 300-residue C-terminal domain . In budding yeast, a homologous kinase is encoded by CDC5 (ref . 3), a gene required for nuclear division late in the mitotic cycle and during meiosis . Murine homologues have also been described . Here we show that the polo gene product immunoprecipitated from extracts of single Drosophila embryos can phosphorylate casein in vitro, and that the kinase activity peaks cyclically at late anaphase/telophase . This contrasts with the cyclical activity of cyclin B-associated p34cdc2 kinase, which is maximal upon entry into mitosis during the rapid cycles of mitosis in the syncytium. Biochem J, 1993 Jun 15, 292 ( Pt 3), 891 - 900 Enzymic characterization of murine and human prohormone convertase-1 (mPC1 and hPC1) expressed in mammalian GH4C1 cells; Jean F et al.; Prohormone convertase-1 (PC1), an endopeptidase that is structurally related to the yeast subtilisin-like Kex2 gene product, has been proposed to be involved in mammalian tissue-specific prohormone processing at pairs of basic residues . To better study this enzyme, a rat somatomammotroph cell line, GH4C1, was infected with vaccinia virus recombinants of murine PC1 (mPC1) and human PC1 (hPC1) . An enzymically active form of each protein was secreted into the cell medium and partially purified by anion-exchange chromatography . The 80-85 kDa enzyme was shown to be Ca(2+)-dependent and exhibited a pH optimum of 6.0 when assayed against a synthetic fluorogenic substrate, acetyl-Arg-Ser-Lys-Arg-4-methylcoumaryl-1-amide . mPC1 and hPC1 displayed identical cleavage selectivity towards a number of fluorogenic substrates, and those incorporating an Arg at the P4 site were most favoured . Synthetic peptides, encompassing the junction between the putative pro-region and the active enzyme, and between the pro-region and the biologically active parathyroid hormone, were shown to be recognized and cleaved specifically at the pair of basic residues by both enzymes . Group-specific proteinase inhibitors such as metal ion chelators and p-hydroxymercuribenzoate, but not phenylmethanesulphonyl fluoride and pepstatin, strongly inhibit the PC1-associated activity . In addition, it is shown that an enzyme activity displaying identical properties is present in the cell medium of uninfected corticotroph AtT-20 cells and that its level is increased following stimulation of secretion by the secretagogue 8-bromo cyclic AMP. Biochem J, 1993 Jun 15, 292 ( Pt 3), 705 - 10 Cloning of a cDNA encoding bovine mitochondrial NADP(+)-specific isocitrate dehydrogenase and structural comparison with its isoenzymes from different species; Huh TL et al.; Mitochondrial NADP(+)-specific isocitrate dehydrogenase (IDP) was co-purified with the pyruvate dehydrogenase complex from bovine kidney mitochondria . The determination of its N-terminal 16-amino-acid sequence revealed that it is highly similar to the IDP from yeast . A cDNA clone (1.8 kb long) encoding this protein was isolated from a bovine kidney lambda gt11 cDNA library using a synthetic oligodeoxynucleotide . The deduced protein sequence of this cDNA clone rendered a precursor protein of 452 amino-acid residues (50,830 Da) and a mature protein of 413 amino-acid residues (46,519 Da) . It is 100% identical to the internal tryptic peptide sequences of the autologous form from pig heart and 62% similar to that from yeast . However, it shares little similarity with the mitochondrial NAD(+)-specific isoenzyme from yeast . Structural analyses of the deduced proteins of IDP isoenzymes from different species indicated that similarity exists in certain regions, which may represent the common domains for the active sites or coenzyme-binding sites . In Northern-blot analysis, one species of mRNA (about 2.2 kb for both bovine and human) was hybridized with a 32P-labelled cDNA probe . Southern-blot analysis of genomic DNAs verified simple patterns of hybridization with this cDNA . These results strongly indicate that the mitochondrial IDP may be derived from a single gene family which does not appear to be closely related to that of the NAD(+)-specific isoenzyme. J Biol Chem, 1993 Jun 15, 268(17), 12682 - 90 The deduced sequence of the novel protransglutaminase E (TGase3) of human and mouse; Kim IG et al.; At least three transglutaminases are involved in terminal differentiation events in the epidermis and its derivatives, such as the hair follicle, presumably in cross-linking structural proteins and in the formation of the cornified cell envelope . Of these, only the transglutaminase 3 is a proenzyme, requiring activation by proteolytic cleavage, and is the least understood . Using oligonucleotides designed from the amino acid sequences of peptides of the guinea pig enzyme, we amplified mRNA and deduced the complete amino acid sequences of the mouse and human protransglutaminase 3 enzymes . Both proteins contain 692 amino acids of molecular mass about 77 kDa . Following expression in yeast, the proenzymes encoded by the full-length cDNA clones are active enzymes and can be further activated 15-fold on treatment with dispase . Although these proteins share 38-53% identity to other members of the transglutaminase family, surprisingly, the mouse, human, and guinea pig enzymes have not been highly conserved and show only 50-75% identity to each other . Much of the sequence variation occurs in the vicinity of the proteolytic activation site which lies at the most flexible and hydrophilic region of the molecule and is flanked by a sequence of 12 residues that are absent from other transglutaminases . We suggest that cleavage of this exposed flexible hinge region promotes a conformational change in the protein to a more compact form, resulting in activation of the enzyme . Expression of mouse and human protransglutaminase 3 mRNAs is regulated by calcium, as for other late differentiation products of the epidermis, suggesting that this enzyme is responsible for the later stages of cell envelope formation in the epidermis and hair follicle. Mol Cell Biochem, 1993 Jun 9-23, 123(1-2), 129 - 38 The function of acyl-CoA-binding protein (ACBP)/diazepam binding inhibitor (DBI); Knudsen J et al.; Acyl-CoA-binding protein has been isolated independently by five different groups based on its ability to (1) displace diazepam from the GABAA receptor, (2) affect cell growth, (3) induce medium-chain acyl-CoA-ester synthesis, (4) stimulate steroid hormone synthesis, and (5) affect glucose-induced insulin secretion . In this survey evidence is presented to show that ACBP is able to act as an intracellular acyl-CoA transporter and acyl-CoA pool former . The rat ACBP genomic gene consists of 4 exons and is actively expressed in all tissues tested with highest concentration being found in liver . ACBP consists of 86 amino acid residues and contains 4 alpha-helices which are folded into a boomerang type of structure with alpha-helices 1, 2 and 4 in the one arm and alpha-helix 3 and an open loop in the other arm of the boomerang . ACBP is able to stimulate mitochondrial acyl-CoA synthetase by removing acyl-CoA esters from the enzyme . ACBP is also able to desorb acyl-CoA esters from immobilized membranes and transport and deliver these for mitochondrial beta-oxidation . ACBP efficiently protects acetyl-CoA carboxylase and the mitochondrial ADP/ATP translocase against acyl-CoA inhibition . Finally, ACBP is shown to be able to act as an intracellular acyl-CoA pool former by overexpression in yeast . The possible role of ACBP in lipid metabolism is discussed. J Mol Biol, 1993 Jun 5, 231(3), 559 - 68 Self-splicing of a Podospora anserina group IIA intron in vitro . Effects of 3'-terminal intron alterations on cleavage at the 5' and 3' splice site; Schmidt U et al.; A shortened derivative of the group IIA intron from the mitochondrial cytochrome-c-oxidase subunit I gene (COI I1) of the ascomycete Podospora anserina can undergo self-splicing in vitro . When compared to self-splicing group IIB introns from yeast mitochondria (aI5c, bI1) the autocatalytic reaction shows a lower efficiency and 5' cleavage takes place predominantly by hydrolysis . In order to test the influence on reaction efficiency and mode of 5' cleavage of the long peripheral structure of domain VI (dVI) we generated mutant Podospora introns that have different structural forms of shortened dVI . Our results show that: (1) in general the size and structure of dVI distal from the branch site is essential for 5' transesterification and influences the efficiency of the second splicing step; (2) 5' transesterification as well as the complete self-splicing reaction is more efficient when the structure of dVI is adapted to that of yeast group IIB introns . Moreover, our data indicate that the postulated gamma-gamma' tertiary interaction is also functional for group IIA introns . A weakening or disruption of this interaction in the Podospora intron leads to a greatly reduced cleavage at the 3' splice site and to a selection of cryptic sites downstream in the 3' exon that almost exclusively restore the strong wild-type gamma-gamma' pairing . The so-called "guide" interaction seems to support the selection of 3' cleavage sites but is of secondary importance in relation to the gamma-gamma' interaction. Gan To Kagaku Ryoho, 1993 Jun, 20(8), 1107 - 21 {Ras p21 and signal transduction}; Kishi K et al.; It is known that ras gene was discovered as a transforming gene of rat sarcoma virus . This gene is conserved ubiquitously from yeast to human, and a dominant activating mutation of ras results in transformation in mammalian cells . Currently it is evident that ras gene regulates cell proliferation and differentiation . The gene product, ras p21, binds to GDP or GTP, and hydrolyzes GTP to GDP and Pi . The GTP-bound form is in active conformation which transmits signals to a downstream target . In this review, we focus on how the activity of ras p21 is regulated and on the mode of action of ras p21 in cell proliferation and differentiation. J Nutr, 1993 Jun, 123(6), 1124 - 8 Selenium requirements of rats for normal hepatic and thyroidal 5'-deiodinase (type I) activities; Vadhanavikit S et al.; The nutritional requirement of selenium for type I 5'-deiodinase activity in thyroid compared with liver was assessed in rats . Male weanling Sprague-Dawley rats were fed a torula yeast-based diet for 20 wk . One group of rats was fed the Se-deficient basal diet (0.01 mg Se/kg) . The other three groups were fed the basal diet plus sodium selenite at 0.05, 0.1 and 0.5 mg Se/kg diet . Liver 5'-deiodinase and glutathione peroxidase (GSH-Px) activities were depressed in the group fed the Se-deficient (basal) diet compared with the other groups . Liver 5'-deiodinase activity in the group fed 0.05 mg Se/kg diet was as high as in the groups fed 0.1 and 0.5 mg Se/kg diet, whereas GSH-Px activities in the groups fed 0.05 and 0.1 mg Se/kg diet were intermediate in value . Feeding the Se-deficient diet for 20 wk did not cause a suppression in 5'-deiodinase in the thyroid, and thyroid GSH-Px activity was approximately 40% of that in the other groups . In rats fed Se-supplemented diets, thyroid GSH-Px was approximately 20% or less of the activity found in liver . Plasma thyroxine was higher in the group fed the Se-deficient (basal) diet, but there were no differences in plasma 3,3',5-triiodothyronine among all groups . The results suggest that the nutritional Se requirement for 5'-deiodinase is less than that for GSH-Px and is approximately 0.05 mg Se/kg in the diet for normal activity in the liver and approximately 0.01 mg Se/kg for normal activity in the thyroid . Thyroid seems to be a priority organ over liver for Se when the intake of the element is limited. Genes Dev, 1993 Jun, 7(6), 1033 - 46 Drosophila 230-kD TFIID subunit, a functional homolog of the human cell cycle gene product, negatively regulates DNA binding of the TATA box-binding subunit of TFIID; Kokubo T et al.; A Drosophila cDNA encoding the largest TFIID subunit (p230) was isolated using a degenerate oligodeoxynucleotide probe based on an amino acid sequence of the purified protein . The entire cDNA sequence contains an open reading frame encoding a polypeptide of 2068 amino acids, corresponding to a calculated molecular mass of 232 kD . The deduced amino acid sequence showed a strong sequence similarity with the protein encoded by a human gene (CCG1) implicated in cell cycle progression through G1, suggesting that p230 may be a target for cell cycle regulatory factors . The recombinant protein expressed in Sf9 cells via a baculovirus vector interacts directly with the TATA box-binding subunit of TFIID (TFIID tau or TBP) from Drosophila, human, and yeast . Surprisingly, recombinant p230 inhibits the TATA box-binding activity and function of TFIID tau, suggesting that p230 interactions with TFIID tau, and possible modulations thereof by other factors may play an important role in TFIID function. Oncogene, 1993 Jun, 8(6), 1593 - 602 The cdk2 kinase is required for the G1-to-S transition in mammalian cells; Tsai LH et al.; In the cell cycle of fission and budding yeast, the p34cdc2/CDC28 kinase is required for both the G1-to-S and G2-to-M phase transitions . In vertebrates, the homologous p34cdc2 kinase is required for G2-to-M phase transitions but appears to be dispensable for DNA synthesis . We have investigated the function of a related kinase, p33cdk2, using serum-stimulated quiescent human fibroblasts . While the p33cdk2 protein was expressed at constant levels throughout the cell cycle, p33cdk2 kinase activity was first detected a few hours prior to the onset of DNA synthesis . Microinjection of anti-p33cdk2 antibodies blocked cells from entering S phase . Pre-adsorption of these antibodies with cdk2 protein abrogated their blocking effect suggesting that the G1 arrest caused by these antibodies is cdk2-specific . These results indicate that p33cdk2 is required for the G1-to-S phase transition in mammalian cells . We also show evidence to suggest that the cyclin E/p33cdk2 complex is likely to be required for entry into S phase since the timing of the cyclin E-associated kinase activity was coincident with that of p33cdk2 and preclearing of either component abolished the majority of the histone H1 kinase activity present in the lysates harvested from the late G1. J Cell Biol, 1993 Jun, 121(5), 1065 - 73 Purification and properties of transgelin: a transformation and shape change sensitive actin-gelling protein; Shapland C et al.; We have purified the transformation and shape change sensitive isoform of an actin associated polypeptide doublet previously described by us (Shapland, C., P . Lowings, and D . Lawson . 1988 . J . Cell Biol . 107:153-161) and have shown that it is evolutionarily conserved as far back as yeast . The purified protein: (a) binds directly to actin filaments at a ratio of 1:6 actin monomers, with a binding constant (Ka) of approximately 7.5 x 10(5) M-1; and (b) causes actin filament gelation within 2 min . Although these activities are controlled by ionic strength (and may be mediated by positively charged amino acid residues) the molecule remains as a monomer irrespective of ionic conditions . EM reveals that the addition of this protein to actin filaments converts them from a loose, random distribution into a tangled, cross-linked meshwork within 1 min, and discrete tightly aggregated foci after 10 min . By use of an "add-back" cell permeabilization system we can rebind this molecule specifically to actin filaments in cells from which it has previously been removed . Since the protein is transformation sensitive and gels actin, we have named it transgelin. Eur J Immunol, 1993 Jun, 23(6), 1277 - 83 Systematic study of human alpha beta T cell receptor V segments shows allelic variations resulting in a large number of distinct T cell receptor haplotypes; Cornelis F et al.; The variation of the alpha beta T cell receptor (TCR) results mainly from rearrangements of germ-line V, D and J elements combined with the processes of N- and P-region addition . In addition to this extensive diversity, diallelic polymorphism is also recognized in V regions of beta loci . Four such polymorphisms have previously been defined, but the full extent of such variation has not yet been established . To investigate allelic polymorphism, we used a strategy based V locus-specific polymerase chain reaction and single-strand conformation polymorphisms . Studying the two V beta 2 loci and the V alpha 8.1 locus, we found that all exhibited a coding polymorphism . One of the V beta 2 loci proved to be the first multiallele segment to be recognized, with three common variants . The second V beta 2 locus, for which none of the two alleles has been identified in cDNA, appeared in fact to be a V beta orphon, in abnormal location on the chromosome 9 . A yeast artificial chromosome containing part of the TCRB locus allowed us to place the first V beta 2 segment on the known map to define haplotypes with two other polymorphic segments: V beta 1 and V beta 6.7 . Multiple distinct haplotypes result from combinations between these polymorphic loci, showing that V beta regions are highly variable between individuals . Two alleles exist at the V alpha 8.1 segment and both are expressed . This represents the first example of a frequent coding polymorphism for TCRA gene . The distribution of allele frequencies for these segments suggest the action of balancing selection . These data add a further dimension to TCR polymorphism and suggest new candidates to explore TCR-encoded susceptibility to autoimmune diseases. Blood, 1993 Jun 1, 81(11), 2854 - 9 Drosophila forkhead homologues are expressed in a lineage-restricted manner in human hematopoietic cells; Hromas R et al.; The forkhead gene (FKH) regulates morphogenesis in Drosophila . It is the prototype of a new family of transcriptional activators . Partially degenerate oligonucleotides to two conserved amino acid sequences of this family were used to prime a polymerase chain reaction (PCR) amplification of HEL cell cDNA . Two unique clones, designated H3 and H8, were isolated that contained homologies to FKH . A third novel clone, 5-3, was isolated by low stringency screening of a chronic myelogenous leukemia cDNA library using H8 as a probe . H3 and 5-3 are preferentially expressed in restricted hematopoietic lineages, while the expression of H8 was ubiquitous . Southern analysis showed that FKH 5-3 is conserved through yeast, which is rare among tissue-specific transcription factors . The H3 and 5-3 clones provide evidence that FKH family members are present in a tissue-restricted manner in humans. J Cell Sci, 1993 Jun, 105 ( Pt 2), 563 - 9 Topoisomerase II inhibition prevents anaphase chromatid segregation in mammalian cells independently of the generation of DNA strand breaks; Clarke DJ et al.; Yeast temperature-sensitive mutants of DNA topoisomerase II are incapable of chromosome condensation and anaphase chromatid segregation . In mammalian cells, topoisomerase II inhibitors such as etoposide (VP-16-123) have similar effects . Unfortunately, conclusions drawn from work with mammalian cells have been limited by the fact that the standard inhibitors of topoisomerase II also generate DNA strand breaks, which when produced by other agents (e.g . ionizing radiation) are known to affect progression into and through mitosis . Here we show that the anti-tumour agent ICRF-193, recently identified as a topoisomerase II inhibitor operating by a non-standard mechanism, generates neither covalent complexes between topoisomerase II and DNA, nor adjacent DNA strand breaks, in mitotic HeLa . However, the drug does prevent anaphase segregation in HeLa and PtK2 cells, with effects similar to those of etoposide . We therefore conclude that topoisomerase II function is required for anaphase chromosome segregation in mammalian cells, as it is in yeast. J Cell Sci, 1993 Jun, 105 ( Pt 2), 481 - 8 Use of a general purpose mammalian expression vector for studying intracellular protein targeting: identification of critical residues in the nuclear lamin A/C nuclear localization signal; Frangioni JV et al.; We have constructed a general purpose mammalian expression vector for the study of intracellular protein targeting . The vector, p3PK, facilitates construction of N- and/or C-terminal fusions of an amino acid sequence of interest to the normally cytosolic protein chicken muscle pyruvate kinase (CMPK) . The vector has been engineered such that any fusion construct can be subcloned into the versatile pJx omega family of mammalian expression vectors and into pGEX bacterial expression vectors, for the generation of affinity reagents . In this paper, we demonstrate the general utility of p3PK by redirecting CMPK to mitochondria (using the twelve amino acid pre-sequence of yeast cytochrome c oxidase subunit IV) and to the nucleus (using a putative eight amino acid nuclear localization signal from human nuclear lamins A and C) . We also report that, contrary to the predictions of previously published work, substitution of a critical residue in the nuclear lamin A/C nuclear localization signal (the equivalent of lysine 128 in the SV40 large T nuclear localization signal) retains nuclear localization, and discuss how amino acid context might affect targeting to the nucleus. J Bioenerg Biomembr, 1993 Jun, 25(3), 233 - 44 A proposed pathway of proton translocation through the bc complexes of mitochondria and chloroplasts; Beattie DS; The cytochrome bc complexes of the electron transport chain from a wide variety of organisms generate an electrochemical proton gradient which is used for the synthesis of ATP . Proton translocation studies with radiolabeled N,N'-dicyclohexylcarbodiimide (DCCD), the well-established carboxyl-modifying reagent, inhibited proton-translocation 50-70% with minimal effect on electron transfer in the cytochrome bc1 and cytochrome bf complexes reconstituted into liposomes . Subsequent binding studies with cytochrome bc1 and cytochrome bf complexes indicate that DCCD specifically binds to the subunit b and subunit b6, respectively, in a time and concentration dependent manner . Further analyses of the results with cyanogen bromide and protease digestion suggest that the probable site of DCCD binding is aspartate 160 of yeast cytochrome b and aspartate 155 or glutamate 166 of spinach cytochrome b6 . Moreover, similar inhibition of proton translocating activity and binding to cytochrome b and cytochrome b6 were noticed with N-cyclo-N-(4-dimethylamino-napthyl)carbodiimide (NCD-4), a fluorescent analogue of DCCD . The spin-label quenching experiments provide further evidence that the binding site for NCD-4 on helix cd of both cytochrome b and cytochrome b6 is localized near the surface of the membrane but shielded from the external medium. J Cell Biol, 1993 Jun, 121(6), 1233 - 43 Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria; Mayer A et al.; Nuclear-encoded proteins destined for mitochondria must cross the outer or both outer and inner membranes to reach their final sub-mitochondrial locations . While the inner membrane can translocate preproteins by itself, it is not known whether the outer membrane also contains an endogenous protein translocation activity which can function independently of the inner membrane . To selectively study the protein transport into and across the outer membrane of Neurospora crassa mitochondria, outer membrane vesicles were isolated which were sealed, in a right-side-out orientation, and virtually free of inner membranes . The vesicles were functional in the insertion and assembly of various outer membrane proteins such as porin, MOM19, and MOM22 . Like with intact mitochondria, import into isolated outer membranes was dependent on protease-sensitive surface receptors and led to correct folding and membrane integration . The vesicles were also capable of importing a peripheral component of the inner membrane, cytochrome c heme lyase (CCHL), in a receptor-dependent fashion . Thus, the protein translocation machinery of the outer mitochondrial membrane can function as an independent entity which recognizes, inserts, and translocates mitochondrial preproteins of the outer membrane and the intermembrane space . In contrast, proteins which have to be translocated into or across the inner membrane were only specifically bound to the vesicles, but not imported . This suggests that transport of such proteins involves the participation of components of the intermembrane space and/or the inner membrane, and that in these cases the outer membrane translocation machinery has to act in concert with that of the inner membrane. Comp Biochem Physiol B, 1993 Jun, 105(2), 345 - 8 Occurrence of a furin-like prohormone processing enzyme in Aplysia neuroendocrine bag cells; Nagle GT et al.; 1 . Strong evidence is accumulating that the endoproteases which process prohormones at dibasic residue cleavage sites are members of a subtilisin-related class of proteases . 2 . Using the polymerase chain reaction (PCR), we have isolated and characterized an Aplysia californica neuroendocrine bag cell cDNA product (270 base pairs) that encodes a sequence which is highly homologous to the subtilisin-related class of processing proteases that includes yeast Kex2, human/mouse/Drosophila furin, human PC2, and mouse PC1/PC3 and PC2 . 3 . The characterized cDNA PCR product showed the highest degree of residue identity with the furin-related group of proteins (human/mouse furin 71%; Drosophila furin 63%) . 4 . These results establish that Aplysia contain a subtilisin-like gene and suggest that the expression of this gene may play a role in processing Aplysia precursor proteins in the bag cells and likely also in the exocrine atrial gland . 5 . Furthermore, the Aplysia nucleotide sequence results, together with available sequence information from human, mouse, and Drosophila furins, provide reasonable evidence that the furin-like enzymes may represent a separate subclass of the subtilisin-like processing enzymes. Hum Mol Genet, 1993 Jun, 2(6), 791 - 6 YAC-assisted cloning of transcribed sequences from the human chromosome 3p21 region; Pengue G et al.; The region surrounding the ZNF35 zinc finger protein gene on 3p21 is of particular interest, as this region of chromosome 3 is frequently involved in rearrangements and/or deletions associated with various human tumors including lung and renal carcinoma . We have analyzed yeast artificial chromosomes (YACs), identified by PCR screening, using oligonucleotides derived from the ZNF35 gene . PFGE and Southern blot/hybridization analysis revealed that the clones cover 750-kb including the ZNF35 gene . The use of specific somatic cell hybrids have allowed us to locate the YAC contig telomeric to the D3F15S2 locus, in a region which is frequently deleted in lung carcinomas . In addition, we have developed a novel cDNA hybridization protocol allowing the isolation of transcribed sequences present in the overlapping YAC clones . Using the cDNA hybridization selection, we have isolated and characterized one transcribed sequence (D3S1362E) from the 3p21 YAC contig and the corresponding cDNA has been isolated . DNA sequencing analysis indicated that the D3S1363E cDNA codes for a putative transcription factor . Northern blot analysis indicated that the D3S1362E sequence hybridized to multiple transcripts in skeletal muscle, and weakly hybridizing transcripts of similar sizes were detected in other tissues. Enferm Infecc Microbiol Clin, 1993 Jun-Jul, 11(6), 299 - 303 {Sexually transmitted diseases in a high risk subpopulation from the province of Soria}; Ulla M et al.; BACKGROUND: Women who practise prostitution constitute a high risk group for acquiring sexually transmitted diseases (STD) . The aim of this study was to know the frequency of these processes among prostitutes in the province of Soria . METHODS: A descriptive transversal study including 86 women who voluntarily went to a Family Planning Center (FPC) in Soria was carried out in the period between October 1986 to December 1991 . Patients were submitted to: investigation of T . vaginalis, N . gonorrhoeae, G . vaginalis, C . trachomatis, S . agalactiae and yeast infections . Complete clinical history was obtained from all including sociodemographic variables and those of risk of infection . RESULTS: Of the 86 women studied 12 (14%) had positive syphilis tests (treponemic and reaginic) . In 12 (14%) N . gonorrhoeae was isolated . The search for C . trachomatis was positive in 16 (18.60%) . In 19 (22.10%) T . vaginalis was directly observed . Twenty-three (26.74%) presented some type of positive serology to B virus . Five (5.81%) were HIV-1 seropositive . Candida spp . was isolated in 27 (31.39%) as was G . vaginalis and S . agalactiae in 21 (24.41%) . Clinical examination did not show any macroscopic lesion suggesting venereal disease . CONCLUSIONS: Infection by Candida spp . and G . vaginalis were found to be the most frequent . The seroprevalence of HIV-1 in this series was the lowest in the country having a clear relation with the use of intravenous drugs (IVDA) . Sexually transmitted diseases maintain a high prevalence in high risk groups such as prostitutes thus requiring energic prevention plans, overall in the group of young IVDA prostitutes. Genomics, 1993 Jun, 16(3), 586 - 92 1.5-Mb YAC contig in Xq28 formatted with sequence-tagged sites and including a region unstable in the clones; Palmieri G et al.; A contig of 20 yeast artificial clones (YACs) has been assembled across 1.5 Mb of Xq28 and formatted with nine previously reported probes and nine STSs developed from the sequence of probes and end fragments of YACs . YAC end fragments were obtained by subcloning, Alu-vector PCR, or primer-ligation PCR methods . Eighteen of the YACs were recovered from a library specific for Xq24-q28; two that fill a gap were obtained from a second library made from total human DNA . One region, containing probes pX78c and 2A1.1, was unstable in YACs, but it was possible to generate a self-consistent map of DNA over the entire contig . Overlaps were confirmed by Southern blot analyses of YAC DNAs, and pulsed-field gel electrophoresis confirmed the extent of the contig and identified at least four CpG islands in the region. Genomics, 1993 Jun, 16(3), 580 - 5 A chromosome 11 YAC library; Qin S et al.; A targeted yeast artificial chromosome (YAC) library for chromosome 11 has been constructed from the J1 cell line that carries a single human chromosome 11 within a hamster DNA background . Interspecies chimeric clones generated during construction of the library were detected during the screening process and eliminated from the library . Contig assembly becomes much less difficult using such a library as the complexity is decreased and the ends of the clone inserts can be rescued for walking to neighboring clones . The library contains > 1824 clones with an average insert length of 337 kb . This represents a fourfold coverage of chromosome 11 or a > 95% chance of recovering a unique single-copy sequence from the library . Two hundred YAC clones were localized by fluorescence in situ hybridization and found to be randomly distributed along the chromosome . The library has been screened with probes for the chromosome 11 markers HBB, GLUR4, H19, and D11S193 . Corresponding YAC clones have been isolated for each locus . This analysis has indicated that the library is unbiased, that cognate YAC clones can be recovered with chromosome 11 markers, and that extensive contig assembly should be feasible. Development, 1993 Jun, 118(2), 401 - 15 Targeted gene expression as a means of altering cell fates and generating dominant phenotypes; Brand AH et al.; We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns . The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers . It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development . We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped . We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells . The GAL4 system can thus be used to study regulatory interactions during embryonic development . In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens . We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway. Plant Physiol, 1993 Jun, 102(2), 623 - 8 Isolation and characterization of cDNAs encoding wheat 3-hydroxy-3-methylglutaryl coenzyme A reductase; Aoyagi K et al.; The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is a key enzyme in the isoprenoid biosynthetic pathway . We have isolated partial cDNAs from wheat (Triticum aestivum) using the polymerase chain reaction . Comparison of deduced amino acid sequences of these cDNAs shows that they represent a small family of genes that share a high degree of sequence homology among themselves as well as among genes from other organisms including tomato, Arabidopsis, hamster, human, Drosophila, and yeast . Southern blot analysis reveals the presence of at least four genes . Our results concerning the tissue-specific expression as well as developmental regulation of these HMGR cDNAs highlight the important role of this enzyme in the growth and development of wheat. Virology, 1993 Jun, 194(2), 537 - 47 Identification of a conserved motif that is necessary for binding of the vaccinia virus E3L gene products to double-stranded RNA; Chang HW et al.; The E3L gene of vaccinia virus encodes the double-stranded (ds) RNA binding proteins p20 and p25 that exhibit inhibitory activity for the IFN-induced, P1/elF-2 alpha protein kinase . A region in the E3L encoded proteins (residues 156-180) shares a high degree of similarity with several proteins that bind double-helical RNA including the P1/elF-1 alpha kinase, bacterial and yeast RNase III, and a human transactivator response element/Rev response element binding protein . In this study, mutants of E3L proteins were constructed in order to determine the region of the proteins required for dsRNA binding and kinase inhibitory activity . Our data indicate that both the region necessary for dsRNA binding and for kinase inhibitory activity are located at the carboxyl terminus of the protein . The E3L proteins with 7 amino acids deleted from the carboxyl terminus (184-190) could bind to dsRNA, but with lower affinity than could the full-length protein . This protein did not detectably inhibit kinase in vitro . Deletion of 26 amino acids from the carboxyl terminus of the E3L proteins (165-190) abolished dsRNA binding activity and kinase inhibitory activity . In addition, mutations at amino acid 164, 167, or 174 severely inhibited binding to dsRNA . On the other hand, deletion of 83 amino acids from the amino terminus did not affect the proteins' ability to bind dsRNA or inhibit kinase . These results suggest that a region of sequence between amino acids 164 and 183 is necessary for E3L proteins' dsRNA binding activity . This region lies within the homologous domain that the E3L proteins share with other dsRNA binding proteins. Biosci Biotechnol Biochem, 1993 Jun, 57(6), 875 - 80 Formation of beta-fructosyl compounds of pyridoxine in growing culture of Aspergillus niger; Suzuki Y et al.; Two pyridoxine compounds were found to be formed in a culture filtrate of Aspergillus niger and A . sydowi, when grown in a medium containing sucrose and pyridoxine . Each of the two compounds I and II was obtained as a white powdered preparation by preparative paper chromatography, gel filtration on Toyopearl HW-40S and Sephadex G-10 columns, DEAE-cellulose column chromatography, and lyophilization . Compounds I and II were identified as 5'-O-(beta-D-fructofuranosyl)-pyridoxine and 5'-O-{beta-D-fructofuranosyl-(2-->1)-beta-D-fructofuranosyl}-pyridoxine, on the basis of the various experimental results, viz., elementary analyses, UV, 1H-, and 13C-NMR spectra, products by hydrolysis with acid and yeast beta-D-fructofuranosidase, migration on paper electrophoresis, and Gibbs reaction in the presence and absence of boric acid . Levansucrase from Microbacterium laevaniformans and yeast beta-D-fructofuranosidase did not catalyze the beta-D-fructofuranosyl transfer from sucrose to pyridoxine to give rise to beta-D-fructofuranosyl-pyridoxine. Hautarzt, 1993 Jun, 44(6), 380 - 4 {Systemic therapy of diffuse effluvium and hair structure damage}; Budde J et al.; A controlled randomized double-blind study was carried out in 72 female patients to compare tolerance and efficacy of two therapeutic agents containing vitamins of the B-group and L-cystine in different compositions versus a placebo in diffuse effluvia and hair structure lesions . Hair swelling as a criterion of hair quality and frontal and parietal anagen rates in trichograms as criteria of hair growth were determined before and after 4 months of therapy . Treatment with active medication 1 was statistically significantly superior to treatment with the placebo according to these criteria . Treatment with active medication 2 was superior to treatment with the placebo but inferior to treatment with active medication 1 . The overall evaluation of efficacy by investigator and patient was in good agreement with these results . The additional active ingredients contained in active medication 1 but not contained in active medication 2 contribute to the efficacy of the medication . They cannot be compensated by the higher amounts of L-cystine contained in active medication 2 . Given their good tolerance, no adverse effects were observed with the two active medications. Science, 1993 May 28, 260(5112), 1338 - 43 Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2; Chardin P et al.; A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene . The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast . A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast . This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro . Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity . Thus hSos1 is a guanine nucleotide exchange factor for Ras . The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro . This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2 . These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1. Nature, 1993 May 27, 363(6427), 371 - 4 Phosphorylation of C-terminal domain of RNA polymerase II is not required in basal transcription; Serizawa H et al.; Phosphorylation of the heptapeptide repeats in the C-terminal domain (CTD) of the largest subunit of RNA polymerase II has been widely proposed as an essential step in transcription initiation on the basis of findings indicating (1) that the CTDs of RNA polymerase II molecules actively engaged in transcription are highly phosphorylated; (2) that polymerase molecules containing non-phosphorylated CTDs preferentially enter the preinitiation complex where they are subsequently phosphorylated; and (3) that essential initiation factors b from yeast, delta from rat, and BTF2(TFIIH) from human cells have closely associated CTD-kinase activities . Here we take advantage of a highly purified enzyme system which supports both CTD phosphorylation and basal transcription to test this hypothesis directly . Using the isoquinoline sulphonamide derivative H-8, which is a potent inhibitor of CTD kinase, we show that basal transcription occurs in the absence of CTD phosphorylation. Nucleic Acids Res, 1993 May 25, 21(10), 2493 - 501 Ancestry and diversity of the HMG box superfamily; Laudet V et al.; The HMG box is a novel type of DNA-binding domain found in a diverse group of proteins . The HMG box superfamily comprises a.o . the High Mobility Group proteins HMG1 and HMG2, the nucleolar transcription factor UBF, the lymphoid transcription factors TCF-1 and LEF-1, the fungal mating-type genes mat-Mc and MATA1, and the mammalian sex-determining gene SRY . The superfamily dates back to at least 1,000 million years ago, as its members appear in animals, plants and yeast . Alignment of all known HMG boxes defined an unusually loose consensus sequence . We constructed phylogenetic trees connecting the members of the HMG box superfamily in order to understand their evolution . This analysis led us to distinguish two subfamilies: one comprising proteins with a single sequence-specific HMG box, the other encompassing relatively non sequence-specific DNA-binding proteins with multiple HMG boxes . By studying the extent of diversification of the superfamily, we found that the speed of evolution was very different within the various groups of HMG-box containing factors . Comparison of the evolution of the two boxes of ABF2 and of mtTF1 implied different diversification models for these two proteins . Finally, we provide a tree for the highly complex group of SRY-like ('Sox' genes), clustering at least 40 different loci that rapidly diverged in various animal lineages. J Biol Chem, 1993 May 25, 268(15), 11389 - 93 High mobility group proteins 14 and 17 can space nucleosomes in vitro; Tremethick DJ et al.; Recently we partially purified from Xenopus laevis ovaries a novel, ATP-dependent, spacing activity that can convert a DNA template consisting of irregularly spaced nucleosomes into a chromatin structure made up of regularly spaced nucleosomes with a repeat length of 160-165 base pairs . In a second independent step, the longer spacing of higher eukaryotic chromatin can be generated by the addition of histone H1 . The partially purified spacing fraction contains several proteins that display chromatographic properties and mobilities on polyacrylamide gels similar to high mobility group (HMG) proteins . For that reason, different HMG proteins were tested for their ability to generate chromatin structures with regularly spaced nucleosomes . In this report, using two different nucleosome assembly systems, we show that the addition of phosphorylated HMGs 14 and 17 to the histone octamer results in the formation of chromatin with a repeat length of 160-165 base pairs . The results are similar to those obtained from studies of chromatin structure in simple cells, such as fungi and yeast, and in active genes. J Biol Chem, 1993 May 25, 268(15), 11222 - 9 A mutational analysis of the polypyrimidine tract of introns . Effects of sequence differences in pyrimidine tracts on splicing; Roscigno RF et al.; The polypyrimidine (py) tract of introns is required for efficient spliceosome assembly and splicing of pre-mRNAs . A detailed mutational analysis of the py tract of an adenovirus 2 intron was carried out . Utilizing a "precursor in pieces" vector system, it was possible to synthesize py tract mutant pre-mRNAs that were otherwise identical . The mutant pre-mRNAs that were otherwise identical . The mutant pre-mRNAs were analyzed for in vitro splicing, for formation of splicing complexes, and for binding to proteins in the HeLa nuclear extract . Chimeric pre-mRNAs that contained the yeast branch point consensus sequence (UAC-UAAC) and altered py tracts were also analyzed . Mutational analysis showed the following . First, any mutation in the py tract that affected splicing did so by interferring with complex A formation in spliceosome assembly . Second, introduction of purines into the py tract is detrimental only if the length of the tract is shortened and if there is a reduction in the number of consecutive uracil residues . Third, uracil and cytosine do not have equivalent functions in the py tract . Our results with chimeric pre-mRNAs also show that a strong py tract can partially replace a weak branch point sequence and a strong branch point sequence can partially replace a weak py tract . Finally, the one surprising finding obtained when examining protein binding was that a mutant pre-mRNA did not bind to heterogeneous nuclear ribonucleoprotein C proteins and yet spliced close to wild type level. Biochemistry, 1993 May 18, 32(19), 5257 - 66 A single-stranded DNA binding protein from Drosophila melanogaster: characterization of the heterotrimeric protein and its interaction with single-stranded DNA; Mitsis PG et al.; We describe the purification to near homogeneity of a single-stranded DNA binding protein from 0-18-h embryos of Drosophila melanogaster . Drosophila SSB (D-SSB) is a heterotrimer with subunits of molecular weight of 70,000, 30,000, and 8000 . It has a Stokes radius of 48.6 +/- 2 A and s20,w = 5.0 +/- 0.2 S . The interaction of D-SSB with ssDNA was examined by the quenching of intrinsic protein fluorescence . The binding site size was determined to be n = 22 +/- 4 nucleotides with a maximum quenching Qm = 35 +/- 3% . Equilibrium titrations indicate that D-SSB binds with low cooperativity, omega = 10-300, and high apparent affinity, K omega = (0.7-5) x 10(7) M-1, at 225 mM NaCl . Sedimentation of D-SSB bound to small oligonucleotides demonstrates that D-SSB does not require protein association for binding . D-SSB stimulates the extent and processivity of DNA synthesis of its cognate DNA polymerase alpha . On the basis of these properties, we conclude that D-SSB is the Drosophila cognate of the human and yeast SSB/RP-A proteins. Proc Natl Acad Sci U S A, 1993 May 15, 90(10), 4567 - 71 Copper-controllable gene expression system for whole plants; Mett VL et al.; We describe a system for gene expression in plants based on the regulation mechanism of the yeast metallothionein (MT) gene . The system consists of two elements: (i) the yeast ace1 (activating copper-MT expression) gene encoding a transcription factor under control of the cauliflower mosaic virus (CaMV) 35S RNA promoter, and (ii) a gene of interest under control of a chimeric promoter consisting of the 90-base-pair domain A of the CaMV 35S RNA promoter linked to the ACE1 transcription factor-binding site . At elevated copper ion concentrations, the ACE1 protein changes conformation, binds to, and activates transcription from the chimeric promoter . To test the functioning of the system in plants, a construct containing the beta-glucuronidase (GUS) reporter gene under control of the chimeric promoter was prepared, and transgenic tobacco plants were produced . It was shown that GUS activity in the leaves of transgenic plants increased up to 50-fold, either after addition of 50 microM CuSO4 to the nutrient solution or after application of 0.5 microM CuSO4 to the plants in a foliar spray . This GUS expression was repressed after the removal of copper ions . The results show that the activity of the described chimeric promoter directly depends on copper ion concentration and that this system can be used in experiments that demand precise timing of expression. J Biol Chem, 1993 May 15, 268(14), 10588 - 92 Structural and functional analyses of the Arg-Gly-Asp sequence introduced into human lysozyme; Yamada T et al.; To determine the functional conformation of the Arg-Gly-Asp (RGD) sequence, we have constructed mutant proteins by inserting 4-12 amino acid residues from the RGD region of human fibronectin between Val74 and Asn75 of human lysozyme . RGDS-, GRGDSP-, TGRGDSPA-, VTGRGDSPAS-, and AVTGRGDS-PASS-introduced mutant lysozymes were expressed in yeast, purified, and designated as RGD4, -6, -8, -10, and -12, respectively . Using baby hamster kidney cells, RGD8, RGD10, and RGD12 were shown to possess high cell adhesion activity nearly equal to 10% of human vitronectin activity . RGD4 and RGD6 exhibited somewhat lower cell adhesion activity . The activities of these mutant proteins were inhibited by the addition of either GRGDSP peptide or polyclonal antibody against vitronectin receptor, as was the case for the vitronectin activity . The results suggest that the cell adhesion signals are transduced to cells through the interaction with the vitronectin receptor . The three-dimensional structures of RGD4 and RGD8 were determined at 1.8-A resolution by x-ray crystallography . A model of the inserted region in RGD4 could be built in the electron density map, but the positions of the preceding residues, Ala73-Val74, were uncertain . The inserted region in RGD8 did not demonstrate continuous electron densities . The results suggest that these RGD sequence-containing regions are highly flexible and that such flexibility could allow the conformation of the RGD regions to be induced to fit into the binding pocket of the integrin receptor. Proc Natl Acad Sci U S A, 1993 May 15, 90(10), 4621 - 5 Translational regulation by the interferon-induced double-stranded-RNA-activated 68-kDa protein kinase; Barber GN et al.; Activation of the interferon-inducible 68-kDa protein kinase (referred to as P68) by double-stranded RNA catalyzes phosphorylation of the alpha subunit of eukaryotic protein synthesis initiation factor 2 . We have analyzed the transient expression of mutant and wild-type kinase molecules in transfected COS cells to examine the effects of the kinase on gene expression in the absence of other interferon-induced gene products . The wild-type P68 kinase was expressed inefficiently whereas a catalytically inactive P68 was expressed at 30- to 40-fold higher levels . Protein stability measurements and primer-extension analysis of human kinase-specific mRNA levels provided evidence that kinase expression was regulated at the level of mRNA translation . Further, cotransfection experiments revealed that the domain II catalytically inactive mutant could stimulate reporter gene protein synthesis in a transdominant manner . We also examined the expression of mutants with deletions in the N-terminal double-stranded RNA binding domains and found that a kinase construct lacking aa 156-243 was expressed at levels comparable to the wild type whereas a P68 construct lacking aa 91-243 was expressed at levels 70-fold higher . Both the inactive domain II P68 mutant and the deletion mutant lacking aa 91-243 were less inhibitory to growth in yeast due to the reduced ability to phosphorylate initiation factor 2 alpha in vivo . In conclusion we have demonstrated that the P68 kinase can regulate mRNA translation primarily of its own mRNA and to a lesser extent of a heterologous mRNA and that this regulation is notably affected by mutations in either the catalytic or N-terminal regulatory domains. J Biol Chem, 1993 May 5, 268(13), 9376 - 80 A novel form of cytochrome P-450 family 4 in human polymorphonuclear leukocytes . cDNA cloning and expression of leukotriene B4 omega-hydroxylase; Kikuta Y et al.; Isolation of cDNA clones for human leukotriene B4 (LTB4) omega-hydroxylase clearly demonstrates that the hydroxylase is a member of the cytochrome P-450 (CYP) superfamily . cDNA clones isolated from a human leukocyte cDNA library with CYP4A4 cDNA as a probe encode a protein of 520 amino acids with a molecular weight of 59,805 . The deduced amino acid sequence contains an invariant cysteine in the conserved heme-binding domain near the C terminus, characteristic of the P-450 superfamily . The microsomes from yeast cells transfected with an expression vector pAAH5 carrying isolated cDNA catalyzed the omega-hydroxylation of LTB4 with a Km value of 0.71 microM, and its activity was significantly inhibited by carbon monoxide and by antisera against CYP4A4, consistent with the properties previously reported with LTB4 omega-hydroxylase in human polymorphonuclear leukocytes . The amino acid sequence of LTB4 omega-hydroxylase (P-450LTB omega) shows 31-44% similarity to those of CYP4A, CYP4B, and CYP4C, whereas less than 25% similarity was observed with any of the other P-450 families . According to the systematic classification of the P-450 superfamily, P-450LTB omega is classified into the CYP4 family but does not belong to any of the known CYP4 subfamilies . This P-450 composes a new subfamily of CYP4 . RNA blot analysis indicated that mRNA hybridized to the cDNA was expressed in the polymorphonuclear leukocytes as well as leukocytes from four individuals . Isolation of the cDNA opens the way to investigate the physiological role and to regulation of the omega-hydroxylase in the inflammation process. Hum Mol Genet, 1993 May, 2(5), 563 - 9 Five skeletal myosin heavy chain genes are organized as a multigene complex in the human genome; Soussi-Yanicostas N et al.; Myosin heavy chain (MyHC) isoforms are encoded by a multigene family in vertebrates . We used genomic DNA mapping by pulse field gel electrophoresis to demonstrate that, in humans, the embryonic, fetal, fast IIB and IIX MyHC genes and a gene coding for a non-identified striated muscle MyHC fast-type isoform (NI), are contained within a 320 kb SalI genomic fragment . The locus is flanked by two CpG islands, separated by 580 kb . In order to further characterize the MyHC genes, a human genomic library constructed in yeast artificial chromosomes (YAC) was screened and five independent clones were isolated . Characterization of these YACs revealed that one of them contains at least five MyHC genes, based on partial sequencing of their conserved third coding exons . Three of these genes correspond to those encoding the embryonic, fetal and fast IIB MyHC isoforms . Moreover, in this YAC clone the embryonic and fetal genes, on the one hand, and the adult fast (IIB, IIX and NI) genes, on the other hand, are contained within two different ClaI fragments . This result suggests that the genes encoding the two developmental forms are adjacent in the human genome and that temporal regulation of the MyHC genes might be related to their organization within the locus . These data represent the first direct evidence for the existence in the human genome of a MyHC multigene locus that contains at least five genes. Genetics, 1993 May, 134(1), 211 - 9 Mapping chromosome rearrangement breakpoints to the physical map of Caenorhabditis elegans by fluorescent in situ hybridization; Albertson DG; A scheme for rapidly mapping chromosome rearrangements relative to the physical map of Caenorhabditis elegans is described that is based on hybridization patterns of cloned DNA on meiotic nuclei, as visualized by fluorescent in situ hybridization . From the nearly complete physical map, DNA clones, in yeast artificial chromosomes (YACs), spanning the rearrangement breakpoint were selected . The purified YAC DNAs were first amplified by degenerate oligonucleotide-primed polymerase chain reaction, then reamplified to incorporate fluorescein dUTP or rhodamine dUTP . The site of hybridization was visualized directly (without the use of antibodies) on meiotic bivalents . This allows chromosome rearrangements to be mapped readily if the duplicated, deficient or translocated regions do not pair with a normal homologous region, because the site or sites of hybridization of the probe on meiotic prophase nuclei will be spatially distinct . The pattern, or number, of hybridization signals from probes from within, or adjacent to, the rearranged region of the genome can be predicted from the genetic constitution of the strain . Characterization of the physical extent of the genetically mapped rearrangements places genetic landmarks on the physical map, and so provides linkage between the two types of map. J Eukaryot Microbiol, 1993 May-Jun, 40(3), 262 - 9 In vitro induced anaerobic resistance to metronidazole in Trichomonas vaginalis; Kulda J et al.; Resistance to metronidazole detectable under anaerobic conditions was induced in two Trichomonas vaginalis strains (TV 10-02 and MRP-2) by cultivation at gradually increasing pressure of the drug (1-100 micrograms/ml) for 12 to 21 months . The resistant derivatives reproduced in anaerobic trypticase-yeast-extract-maltose medium at 100 micrograms/ml metronidazole and showed very high values of minimal lethal concentration for metronidazole in anaerobic in vitro assays (556-1,600 micrograms/ml at 48-h exposure to the drug) . Stepwise selection was necessary to develop the resistance in either strain . Attempts to induce resistance by prolonged maintenance of trichomonads with constant, low or moderate drug concentrations (3-10 micrograms/ml) were unsuccessful . Freshly developed resistance to high concentrations of metronidazole was unstable in absence of drug pressure as well as after cryopreservation . Development of stable resistance required further cultivation at 100 micrograms/ml metronidazole . Unstable substrains did not revert to original susceptibility . They retained a moderate level of resistance, being able to grow at 10 micrograms/ml metronidazole . The strains with fully developed resistance had no activity of the hydrogenosomal enzymes pyruvate: ferredoxin oxidoreductase and hydrogenase and ceased uptake of {14C}-metronidazole . These findings indicate that the pyruvate oxidizing pathway responsible for metronidazole activation was inactivated and metabolism of the drug stopped. Plant Foods Hum Nutr, 1993 May, 43(3), 191 - 6 Effect of processing on the mycoflora and aflatoxin B1 level of a cassava-based product; Adegoke GO et al.; Cassava bread was prepared by pre-gelling, battering and baking cassava flour to which were added, in moderate amounts, sugar, yeast solution and edible oil . Baking was at 215 degrees C for 40 min . No mould was isolated from the cassava bread and the mean value of aflatoxin B1 (AFB1) for the three subsamples of cassava bread was 0.03 microgram/kg . The cassava tuber (Manihot esculenta Crantz), which was used for the production of cassava bread had an initial AFB1 level of 1.91 micrograms/kg and the dominant mycoflora were Penicillium oxalicum, Aspergillus flavus, Fusarium spp and some unidentified fungi. Mol Pharmacol, 1993 May, 43(5), 827 - 32 Major pathway of imipramine metabolism is catalyzed by cytochromes P-450 1A2 and P-450 3A4 in human liver; Lemoine A et al.; The metabolism of imipramine by human liver microsomes was examined using a combination of five strategies . Human hepatic microsomes produced N-desmethylimipramine (84%), 2-hydroxyimipramine (10%), and 10-hydroxyimipramine (6%) . Preincubation of human hepatocytes in culture with beta-naphthoflavone and macrolides exclusively induced the formation of desmethylimpramine (552%, p < 0.05, and 234%, p < 0.003, respectively) . Correlations were obtained between rates of imipramine demethylation and cytochrome P-450 (P-450) 1A2 (r = 0.88, p < 0.001) and P-450 3A (r = 0.80, p < 0.02) concentrations in human liver microsomal preparations from 13 different subjects . Anti-P-450 1A2 and anti-P-450 3A antibodies selectively inhibited N-demethylation (80% and 60%, respectively) . N-Demethylation was completely inhibited when anti-1A2 and anti-3A were added simultaneously . Kinetic studies with human microsomes confirm the contribution of two different enzymes in the N-demethylation . The Km of 1A2 was similar to the high affinity Km in human liver microsomes, whereas the Km of 3A was similar to the low affinity Km in human liver microsomes . P-450 1A2 was apparently more efficient than 3A4 (lower Km and higher Vmax) but was expressed in much lower concentration . Human P-450s 1A2 and 3A4 expressed in yeast efficiently produced desmethylimipramine . These results suggest that P-450 1A2 and P-450 3A4 are the major enzymes involved in imipramine N-demethylation in human hepatic microsomes . Similar experiments were conducted using P-450 2D6, and they confirmed that P-450 2D6 catalyzes imipramine 2-hydroxylation . Interindividual variations in 3A4 hepatic content may explain the large variations in imipramine blood levels observed after uniform dosages and thus may explain the variations in clinical efficacy . Caution might be advised in the clinical use of tricyclic antidepressants when drugs are also administered that induce or inhibit P-450s 3A4 and 1A2. Genes Dev, 1993 May, 7(5), 844 - 56 An ATP-dependent inhibitor of TBP binding to DNA; Auble DT et al.; An activity in yeast nuclear extracts (termed ADI) is described that inhibits the binding of the TATA-binding protein (TBP) to DNA in an ATP-dependent manner . The effect is reversible, ATP specific, rapid, and is not promoter specific . ADI is specific for TBP because three other protein-DNA complexes are not affected by ADI . The action of ADI is blocked by association of TFIIA with the TBP-DNA complex . ADI activity at the adenovirus major late promoter requires a segment of DNA upstream from the TATA sequence, suggesting that ADI recognizes aspects of both TBP and DNA . The evolutionarily conserved carboxy-terminal domain of TBP is sufficient for ADI recognition, and amino acids in the basic region of TBP are required for ADI action . ADI can repress transcription in vitro in an ATP-dependent manner . In the presence of ADI, both TFIIA and TBP are required to commit a template to transcription . A model of ADI action is proposed, and possible roles of ADI in the regulation of the transcription complex assembly are discussed. Oncogene, 1993 May, 8(5), 1149 - 60 Chimeric c-Jun containing an heterologous homodimerization domain transforms primary chick embryo fibroblasts; Castellazzi M et al.; To investigate a possible role for c-Jun homodimers in c-Jun-mediated transformation, we designed two chimeric c-Jun derivatives, called c-Juneb1 and c-Jungcn4 . In these chimeric derivatives the natural dimerization domain of c-Jun was replaced by the heterologous homodimerization domain of the Epstein-Barr virus EB1 or the yeast GCN4 transcription factor . Chick embryo fibroblasts chronically infected with retroviruses expressing c-Jun, c-Juneb1 or c-Jungcn4 are transformed . Infection with each construction results in sustained growth in low serum and development of colonies from single cells in agar with similar efficiencies . In contrast to c-Jun, c-Juneb1 and c-Jungcn4 confer additional phenotypic alterations related to in vitro transformation including a condensed cell morphology and ability to develop highly invasive, fast growing colonies in agar . These data suggest that c-Jun homodimers can transform chick embryo fibroblasts and activate cellular functions which influence cell morphology and invasive potential in agar . These findings are consistent with the notion that cellular transformation by c-jun is mediated by c-Jun homodimers. J Cell Sci, 1993 May, 105 ( Pt 1), 1 - 5 A novel member of the dynamin family of GTP-binding proteins is expressed specifically in the testis; Nakata T et al.; Dynamin is a member of a new GTPase family, which includes the mouse Mx protein, the yeast VPS1 and the Drosophila shibire gene product . A high homology with the shibire product suggests a role for dynamin in the endocytotic process, but it is expressed only in mature neurons . We identified two additional dynamin-like proteins in rats, by using the polymerase chain reaction with degenerate primers corresponding to the GTP-binding areas conserved between dynamin and VPS1 . The full coding sequence of one of them, dynamin-2, revealed that it has 848 amino acids and has great similarity with brain dynamin and the shibire product . Northern blot analysis and in situ hybridization revealed its expression to be specific to the seminiferous tubules in the testis . Dynamin-2 (testis type dynamin) was expressed in germ-cell-depleted testis as well, indicating its expression in Sertoli cells . Our data imply that a number of dynamin family proteins, which are products of distinct genes, may play different roles specific to each cell type in the same rat. Eur J Clin Microbiol Infect Dis, 1993 May, 12(5), 336 - 42 Evaluation of five commercial antifungal susceptibility testing systems; Druetta A et al.; Five commercial antifungal susceptibility testing systems were studied for repeatability and reproducibility as well as concordance of results with the MICs for ten reference strains belonging to six different species . Repeatability was determined by testing each strain in triplicate on the same day, and reproducibility by repeating this triple determination on three different days . On the basis of 630 yeast-antifungal agent results for Mycototal and Mycostandard, 540 for Candifast, and 450 for ATB Fungus and Diff Test, repeatability was consistently equal to or greater than 95% . Reproducibility was 80.07% for Candifast and greater than 95% for the other systems . The concordance with the reference MICs was 51.65% for Candifast, 75.33% for ATB Fungus, 80.89% for Diff Test, 90.16% for Mycostandard and 90.32% for Mycototal . Although the performance of Diff Test and ATB Fungus was satisfactory, Mycototal and Mycostandard gave notably better results with imidazoles . Mycostandard, which is easier to use and includes tests for fluconazole and itraconazole, would seem to be potentially the most useful antifungal susceptibility test available at present. Am J Otolaryngol, 1993 May-Jun, 14(3), 199 - 205 Histoplasmosis of the larynx; Sataloff RT et al.; INTRODUCTION: Laryngeal histoplasmosis was first described in 1952 . Since then, fewer than 100 cases had been reported . This dimorphic fungus is endemic in the Mississippi and Ohio River Valleys . The yeast phase is responsible for human infection . METHODS: We report a 44-year-old woman who developed laryngitis . The markedly abnormal larynx, which could have been mistaken for papillomatosis, was biopsied, at which time the diagnosis of histoplasmosis was confirmed . Treatment with oral ketoconazole was instituted . RESULTS: Objective voice assessment showed abnormalities of maximum phonation time, speaking fundamental frequency, perturbation, percent voicing, mean flow rate, and spectral pattern . Subsequent to antifungal therapy, objective measures were improved . CONCLUSION: This represents the first case of laryngeal histoplasmosis in which response to therapy is documented by objective vocal assessment. Somat Cell Mol Genet, 1993 May, 19(3), 265 - 74 Neurofibromatosis type 1 gene product (neurofibromin) associates with microtubules; Gregory PE et al.; The neurofibromatosis type 1 (NF1) gene was recently identified by positional cloning and found to encode a protein with structural and functional homology to mammalian and yeast GTPase-activating proteins (GAPs) . Using antibodies directed against the NF1 gene product, a protein of approximately 250 kDa was identified and termed neurofibromin . Double-indirect immunofluorescent labeling with anti-neurofibromin and anti-tubulin antibodies demonstrates that neurofibromin associates with cytoplasmic microtubules . Immunoblotting of microtubule-enriched cytoplasmic fractions, using antibodies generated against neurofibromin, shows that neurofibromin copurifies with microtubules . When portions of neurofibromin are expressed in Sf9 insect cells they associate with polymerized microtubules; furthermore, the critical residues for this interaction reside within the GAP-related domain of neurofibromin . The unexpected association of neurofibromin with microtubules suggests that neurofibromin is involved in microtubule-mediated intracellular signal transduction pathways. Pediatr Infect Dis J, 1993 May, 12(5), 438 - 45 Pediatric experience with recombinant hepatitis B vaccines and relevant safety and immunogenicity studies; Greenberg DP; Yeast-derived recombinant hepatitis B vaccines have replaced plasma-derived vaccines in the United States and have now been given to millions of infants and children throughout the world . Routine immunization of infants in the United States with hepatitis B vaccine has been endorsed as the optimal means to prevent infection . The recombinant vaccines have an excellent safety record; most children have no adverse reactions whereas a few experience only minor local and systemic reactions that resolve within a short time . Both of the vaccines licensed in the United States are highly immunogenic in infants and children who complete a three dose vaccination sequence . Approximately 95 to 100% achieve protective levels of antibody to hepatitis B surface antigen (> or = 10 mIU/ml) after three doses . Immunization may begin at birth or at 1 to 2 months of age, and hepatitis B vaccine may be given simultaneously with other routine childhood vaccines . Antibody levels to hepatitis B surface antigen gradually wane over time, and the duration of maintaining protective levels correlates strongly with the peak level achieved . The protective efficacy against perinatal transmission from mothers who are positive for hepatitis B surface antigen and e antigen is 90 to 100% when the first dose of vaccine is administered at birth with hepatitis B immunoglobulin . In highly endemic populations immunization in infancy also protects against horizontal transmission from chronically infected family members . Studies currently in progress will determine the duration of protection, the potential need for booster doses and the feasibility of combining antigens in multivalent vaccines. J Microsc, 1993 May, 170 ( Pt 2), 167 - 71 Electron microprobe study of the effect of long-term vacuum storage of freeze-dried cryosections on distribution of elements in cells; Pelc R et al.; The distribution of elements in yeast cells was measured on freshly prepared, freeze-dried cryosections and compared with the distribution obtained on the same sections after storage for 20 months in a vacuum below 2.6 kPa . The average concentration of phosphorus remained unchanged but was equalized throughout the cells, i.e . it migrated from vacuoles into the cytoplasm and mitochondria . Zinc remained preferentially localized in the vacuoles, but the ratio between vacuolar and cytoplasmic zinc concentrations decreased about three-fold . Cytoplasmic and mitochondrial concentrations of potassium remained unchanged while partial release from the vacuoles and subsequently from the cells was observed . This resulted in a homogeneous distribution of potassium in the cells after 20 months and some of the vacuolar potassium appeared in spectra of formvar film measured several micrometres from the cells . A large increase in the sodium (from 160 to 360% more than in fresh sections), magnesium (from 110 to 200% more) and sulphur (from 70 to 350% more) contents was observed in all cellular compartments (except for vacuoles, where only a 20% increase in the magnesium content was observed), while chlorine was almost completely released from the cells . The limitations of the use of long-term vacuum-stored cryosections for electron microprobe analysis of cells are discussed. Curr Genet, 1993 May-Jun, 23(5-6), 501 - 7 Isolation and nucleotide sequence of the 5-aminolevulinate synthase gene from Aspergillus nidulans; Bradshaw RE et al.; The structural gene for 5-aminolevulinate (ALA) synthase has been cloned and sequenced from the filamentous fungus Aspergillus nidulans using an oligonucleotide probe based on a highly conserved-amino-acid sequence found in ALA synthase genes of a wide range of species . The cloned gene, hemA, has a 5' untranslated mRNA of 92 nucleotides (nt) and one intron (64 nt) . The deduced protein sequence (648 amino acids) shows 64% identity to the yeast ALA synthase in the C-terminal region of 453 amino acids . The N-terminal region is typical of ALA synthase proteins in that the specific amino-acid sequence is not conserved but consists of a "leader" region rich in basic amino acids, believed to be involved in mitochondrial targeting, followed by a stretch of largely hydrophobic residues which may allow interaction with the inner mitochondrial membrane . Under the conditions used the transcription of hemA was unaffected by dextrose repression, heat shock, or oxygen levels. Agents Actions, 1993 May, 39(1-2), 69 - 71 In vitro activation of hepatic glutathione reductase from mice by lobenzarit disodium; Armesto J et al.; Glutathione reductase activity from mice liver is significantly enhanced by lobenzarit disodium at concentrations between 0.3 and 1.5 mM . A maximum activation of almost 30% is achieved at a drug concentration of 0.9 mM . Similar results were observed with glutathione reductase from human leukocytes, but not with the enzyme from yeast . By preincubation with the enzyme from mice liver, lobenzarit also proved to prevent, at least partially, the immediate inhibition caused by the well-known thiol-reacting agents, thus indicating a protecting effect on the catalytically important thiol residue of the enzyme . The results here obtained explain in part the recently found hepatoprotective effect of lobenzarit disodium against acute liver toxicity induced by acetaminophen in mice. Cytokine, 1993 May, 5(3), 213 - 23 Expression and characterization of bioactive recombinant ovine TNF-alpha: some species specificity in cytotoxic response to TNF; Green IR et al.; We have expressed and partially purified recombinant ovine tumour necrosis factor alpha (rovTNF-alpha) using a yeast Ty, virus like particle, expression system . RovTNF-alpha is at least as active as recombinant human TNF-alpha (rhTNF-alpha) in two different bio-assays performed on ovine material, whilst approximately 1000-fold more rovrTNF-alpha than rhTNF-alpha is required to induce the same level of cytotoxicity in TNF-sensitive murine cell lines L929 and WEHI 164 clone 13 . When cytotoxic assays are performed on the porcine TNF sensitive cell line PK(15)-1512, rovTNF-alpha shows about 2 logs greater activity than on murine cells, whilst rhTNF-alpha is about 1 log more active . A monoclonal antibody, raised against rovTNF-alpha, has been used to demonstrate the presence of nanogram amounts of an appropriately sized glycoprotein to be native ovine TNF-alpha in supernants of LPS stimulated ovine alveolar macrophages . These samples show no detectable cytotoxicity to L929 cells, although they show activity attributable to TNF-alpha (through neutralization by a polyclonal antiserum raised to rovTNF-alpha) in an assay on ovine material . The relative lack of activity on murine cells helps to explain previous reports of inability to assay native ovine TNF-alpha using these cells, in spite of their routine use to assay TNF-alpha from several other species . The sequence features in ovine TNF-alpha which might reduce its affinity for the murine TNF type 1 receptor are discussed. Drug Metab Dispos, 1993 May-Jun, 21(3), 403 - 9 Oxidation of the antihistaminic drug terfenadine in human liver microsomes . Role of cytochrome P-450 3A(4) in N-dealkylation and C-hydroxylation; Yun CH et al.; The antihistaminic drug terfenadine, alpha-{4-(1,1-dimethylethyl)phenyl}-4-(hydroxydiphenylmethyl)-1- piperidinebutanol (Seldane), is of interest because of its lack of sedative properties . Major routes of metabolism include oxidative N-dealkylation to 4-(hydroxydiphenylmethyl)-piperidine (1) and oxidation of a tert-butyl methyl group to a primary alcohol (2), which is subsequently oxidized to a carboxylic acid . Rates of formation of 1 and 2 varied approximately 30-fold in the 17 human liver microsomal samples examined and were highly correlated with each other, suggesting that the same enzyme may be involved in both oxidations . The rates of formation of 1 and 2 were both correlated with rates of nifedipine oxidation (a marker of cytochrome P-450 (P-450) 3A4) but not with markers for other human P-450s . Microsomal oxidation of (both enantiomers of) terfenadine to 1 and 2 was markedly inhibited by gestodene, a selective mechanism-based inactivator of P-450 3A enzymes but not by any of several other P-450 inhibitors . Antibodies raised against P-450 3A4 could inhibit most of the oxidation of (both enantiomers of) terfenadine to 1 and 2 in a microsomal sample having high catalytic activity but antibodies recognizing other P-450s had no effect . The oxidation of terfenadine to 1 and 2 was catalyzed by purified human liver microsomal P-450 3A4 and by partially purified yeast recombinant P-450 3A4 . These results provide evidence that P-450 3A4 (and possibly other P-450 3A enzymes) play a major role in the oxidation of (both enantiomers of) terfenadine to both of its major oxidation products.(ABSTRACT TRUNCATED AT 250 WORDS) Mech Dev, 1993 May, 41(2-3), 155 - 61 Dpbx, a new homeobox gene closely related to the human proto-oncogene pbx1 molecular structure and developmental expression; Flegel WA et al.; Recently, a new class of homeodomain containing proteins, pbx1, pbx2, and pbx3 has been described . pbx proteins are most closely related to two yeast regulatory proteins, a1 and alpha 2 . Here, we identify and characterize the pbx homolog in Drosophila, designated Dpbx . Dpbx is 95% identical to the pbx proteins within the homeodomain and, more remarkably, is 85% to 88% identical within a 201 amino acid region adjacent to the homeodomain . Cytologically, the Dpbx gene is located on the X chromosome at 14A . mRNA expression is both maternal and zygotic and occurs throughout the life cycle . Prior to full germband retraction, Dpbx is rather ubiquitously present and variations are minor . The most notable feature of Dpbx expression is that after germband retraction, high levels of Dpbx are observed in the anterior portion of the ventral nerve cord. Mol Gen Genet, 1993 May, 239(1-2), 145 - 57 Chromosome walking with YAC clones in Arabidopsis: isolation of 1700 kb of contiguous DNA on chromosome 5, including a 300 kb region containing the flowering-time gene CO; Putterill J et al.; The co mutation of Arabidopsis thaliana causes a late-flowering phenotype that is insensitive to day-length . The mutation was mapped previously to the upper arm of chromosome 5, approximately 1.6 cM from the chalcone synthase gene (CHS) . We were provided with five yeast artificial chromosome (YAC) libraries and used these to perform a chromosome walk from CHS to the CO gene . In this paper we report the isolation of 1700 kb of contiguous Arabidopsis DNA, which represents approximately 1%-2% of the genome, inserted in YACs . This required the detailed analysis of 67 YACs, from which 87 end probes were isolated and examined in hybridisation experiments . This analysis showed that approximately 40% of the YACs presented problems in chromosome walking experiments because they contained repetitive sequence at one of their termini, were chimaeric or because part of the plant DNA was deleted . DNA fragments isolated from YACs were used as restriction fragment length polymorphism (RFLP) markers to localize CO to a 300 kb region within the cloned DNA . We compare the physical distance between CHS and CO with the genetic distance and find that in this region 1 cM is equivalent to approximately 200 kb. New Horiz, 1993 May, 1(2), 202 - 13 Pathogenesis and management of Candida infection syndromes in non-neutropenic patients; Solomkin JS; Infections due to Candida spp . are increasingly common . Despite a number of studies examining the characteristics of fungemic (non-neutropenic) patients, there are no prospective studies defining indications for therapy before the appearance of fungemia . This article reviews the potential pathways for dissemination of Candida from the gastrointestinal tract, believed to be the primary entry route for yeast . Particular attention is focused on mucosal shedding at the villous tip and the potential role of microfold cells in this process . Therapeutic usage of amphotericin B, the agent of choice for serious Candida infection, is reviewed, along with usage of 5-flucytosine and fluconazole. Pediatr Res, 1993 May, 33(5), 458 - 65 Studies of immunomodulating actions of RNA/nucleotides . RNA/nucleotides enhance in vitro immunoglobulin production by human peripheral blood mononuclear cells in response to T-dependent stimuli; Jyonouchi H et al.; We have shown previously that yeast RNA preparations enhance in vitro antibody and Ig production to T-dependent antigens in normal B6 mice . In this study, Ig production by human peripheral blood mononuclear cells from adult volunteers was examined under RNA and mononucleotide-supplemented culture conditions . RNA significantly enhanced IgM and IgG production in a dose-dependent manner to pokeweed mitogen, T-dependent stimuli, and keyhole limpet hemocyanin modified with trinitrophenol, T-dependent antigens . IgM and IgG production in response to T-independent stimuli were not significantly altered by RNA and mononucleotides . IgA production appeared not to be influenced by RNA and mononucleotides irrespective of the stimuli provided . Interestingly, spontaneous IgM production also appeared to be enhanced by RNA . When RNA was degraded, oxidized, or decomposed of pyrimidine bases, this enhancing action of RNA on Ig production was considerably reduced . RNA was most effective when present from d 0 of the culture . Its enhancing action was lost when it was added at d 3 of the culture or later, when T cells were depleted, or when direct interactions between T cells and non-T cells were not permitted in the culture . Thus, RNA may enhance IgM and IgG production by human peripheral blood mononuclear cells to T-dependent stimuli partly by influencing the process of direct cellular interactions between T and non-T cells in the early stage of B-cell activation. Biochem Biophys Res Commun, 1993 Apr 30, 192(2), 849 - 53 The primary structure of rat ribosomal protein L36; Chan YL et al.; The amino acid sequence of the rat 60S ribosomal subunit protein L36 was deduced from the sequence of nucleotides in a recombinant cDNA . Ribosomal protein L36 has 104 amino acids, the NH2-terminal methionine is removed after translation of the mRNA and has a molecular weight of 12,128 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 8 to 11 copies of the L36 gene . The mRNA for the protein is about 500 nucleotides in length . Rat L36 is related to yeast ribosomal protein YL39. Biochem Biophys Res Commun, 1993 Apr 30, 192(2), 583 - 9 The primary structure of rat ribosomal protein L29; Chan YL et al.; The amino acid sequence of the rat 60S ribosomal subunit protein L29 was deduced from the sequence of nucleotides in a recombinant cDNA . Ribosomal protein L29 has 155 amino acids, the NH2-terminal methionine is removed after translation of the mRNA, and has a molecular weight of 17,183 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 20 to 22 copies of the L29 gene . The mRNA for the protein is about 750 nucleotides in length . Rat L29 is related to yeast ribosomal protein YL43. Nucleic Acids Res, 1993 Apr 25, 21(8), 1797 - 804 Domain 5 interacts with domain 6 and influences the second transesterification reaction of group II intron self-splicing; Dib-Hajj SD et al.; The role of domain 5 (d5) from the self-splicing group II intron 5 gamma of the COXI gene of yeast mitochondrial DNA in branching and 3' splice site utilization has been studied using a substrate transcript lacking d5 (delta d5 RNA) . This RNA is completely unreactive in vitro, but releases 5' exon by hydrolysis under various reaction conditions when d5 RNA is added in trans . Under an extreme reaction condition, some accurate branching and splicing occur . Much more efficient use of a 3' splice site is obtained when delta d5 RNA is complemented by a transcript containing the wild-type domains 5 and 6 plus the 3' exon . While most delta d5 RNA molecules in that protocol still react by hydrolysis at the 5' splice site, the branching that occurs uses only the d6 tethered to d5 that is provided in trans . The use of this d6 and the 3' splice site also linked to d5, along with the observed indifference to the other d6 and 3' splice site resident in the delta d5 RNA, indicates that d5 plays a key role in positioning d6 for the first reaction step as well as in 3' splice site use . Two models for the manner by which d5 interacts with d6 are discussed. J Biol Chem, 1993 Apr 25, 268(12), 8422 - 4 Evolution of the type II hexokinase gene by duplication and fusion of the glucokinase gene with conservation of its organization; Kogure K et al.; The type I, II, and III isozymes of mammalian hexokinase (100 kDa) all consist of a duplicated, highly homologous peptide sequence, each half of which is very similar to that of glucokinase (type IV hexokinase, 50 kDa) and yeast hexokinase (50 kDa) . We isolated a genomic clone of type II hexokinase that contained five exons encoding the C-terminal region of type II hexokinase . The positions of intron insertions of the isolated clone were found to be identical with those of the glucokinase gene, indicating that the type II hexokinase gene arose from the glucokinase gene . Furthermore, we prepared two DNA fragments of the type II hexokinase gene amplified from total genomic DNA . The exons in these fragments were found to be constructed by linkage of the coding region of the last exon and second exon of the glucokinase gene, indicating that the hexokinase gene arose by fusion of two glucokinase genes . These results clearly show that the mammalian hexokinase gene evolved from the glucokinase gene by gene duplication and fusion with conservation of the gene organization. Science, 1993 Apr 23, 260(5107), 536 - 9 Negative regulation of G1 in mammalian cells: inhibition of cyclin E-dependent kinase by TGF-beta; Koff A et al.; Transforming growth factor-beta (TGF-beta) is a naturally occurring growth inhibitory polypeptide that arrests the cell cycle in middle to late G1 phase . Cells treated with TGF-beta contained normal amounts of cyclin E and cyclin-dependent protein kinase 2 (Cdk2) but failed to stably assemble cyclin E-Cdk2 complexes or accumulate cyclin E-associated kinase activity . Moreover, G1 phase extracts from TGF-beta-treated cells did not support activation of endogenous cyclin-dependent protein kinases by exogenous cyclins . These effects of TGF-beta, which correlated with the inhibition of retinoblastoma protein phosphorylation, suggest that mammalian G1 cyclin-dependent kinases, like their counterparts in yeast, are targets for negative regulators of the cell cycle. Biochem Biophys Res Commun, 1993 Apr 15, 192(1), 119 - 23 Onset of new catalytic activity in immobilized spores of Aspergillus ochraceus TS due to in situ germination: C17-C20 lysis accompanies 11 alpha-hydroxylation of steroid; Dutta TK et al.; The change in product pattern during transformation of progesterone by calcium alginate entrapped spores of Aspergillus ochraceus TS (A . ochraceus TS) due to germination in situ is reported . While progesterone was transformed exclusively to its 11 alpha-hydroxy derivative by both free and immobilized spores of A . ochraceus TS, the latter germinated in situ by yeast extract furnished 11 alpha-hydroxyprogesterone and C19 steroids during transformation under identical conditions . The characterization of metabolite(s) and the pathway proposed demonstrated that delta 1-dehydrogenation and lysis of C17-C20 bond are apparently two independent reactions . The change in product profile due to activation of immobilized spores is believed to be caused by accumulation of compatible solutes in the biocatalyst which had relatively low water content. Biochemistry, 1993 Apr 13, 32(14), 3527 - 34 Association of the A subunits of recombinant placental factor XIII with the native carrier B subunits from human plasma; Radek JT et al.; Interactions of a recombinant human placental protein (rA2) expressed in yeast and considered to be identical to the catalytic A2 subunits of factor XIII, the fibrin stabilizing factor zymogen, were examined with the native carrier subunits (B2) of the factor isolated from human plasma . Nondenaturing electrophoresis and HPLC gel-filtration experiments showed a tight binding of rA2 to B2 for forming an ensemble similar to that of plasma factor XIII (A2B2) . In the presence of excess B2, however, some higher ordered oligomers (rA2Bn, where n > 2) were also seen in electrophoresis . The same technique revealed a microheterogeneity in the rA2 preparation; nevertheless, all isoforms could bind to B2 . By employing an ELISA procedure for measuring free B2 in mixtures with rA2, an apparent binding constant of 4 x 10(7) M-1 was derived for the association of rA2 with B2 . Fluorescence depolarization was used to monitor the heterologous association of rA2 with fluorescein-labeled B2F as well as the dissociation of the rA2B2F structure . The former was characterized by an increase, and the latter by a decrease, in the fluorescence anisotropy of the system . Binding of rA2 to B2F (pH 7.5, mu = 0.315, 37 degrees C) was not influenced by low concentrations of Ca2+ (< or = 30 mM), and rA2B2F proved to be quite stable under these conditions . Much higher concentrations of Ca2+, as well as higher ionic strengths, were required to dissociate this assembly . By contrast, release of B2F from the thrombin-modified rA2'B2F occurred rapidly in the presence of low concentrations of Ca2+ at low ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1993 Apr 12, 320(3), 215 - 8 Positional and additive effects of basic amino acids on processing of precursor proteins within the constitutive secretory pathway; Watanabe T et al.; We have recently shown that the Arg/Lys-X-Lys/Arg-Arg or Arg/Lys-X-X-X-Lys/Arg-Arg sequence serves as a signal for cleavage of precursor proteins within the constitutive secretory pathway, and this cleavage is catalyzed by furin, a mammalian homolog of the yeast Kex2 protease . In this study, we further examined sequence requirements for the constitutive precursor cleavage . Based on the data concerning cleavage efficiencies of various prorenin mutants with amino acid substitution(s) around the native cleavage site expressed in CHO cells, we revised the sequence rules that govern the constitutive cleavage as follows: (i) the Arg residue at position -1 is essential; (ii) in addition to the Arg at position -1, at least two out of the three basic residues at positions -2, -4, and -6 are required for efficient cleavage (the presence of all the three basic residues results in most efficient cleavage); (iii) at position +1, a hydrophobic aliphatic amino acid is not suitable. BMJ, 1993 Apr 10, 306(6883), 947 - 8 Where are we now with vaccines against AIDS? Schild GC, Stott EJ. PIP: Extensive studies in experimental animal models and phase I and II clinical trials in humans provide some grounds for optimism about developing a vaccine for AIDS . Over 200 monkeys have been protected by simple inactivated vaccines against infection with a lethal challenge dose of simian immunodeficiency virus (SIV) . Passive transfer of the antibody to SIV has been shown to prevent infection . Recent vaccination with attenuated, live SIV protected against a challenge with 1000 monkey infectious doses of virus . Studies in chimpanzees have been limited to the unrepresentative IIIB strain of HIV-1 . Purified recombinant envelope protein either as the gp 120 surface unit or as the entire gp 160 protein has protected a proportion of chimpanzees against infection . Several experimental immunogens have been tested in phase I and II clinical trials in human volunteers . 5 different recombinant HIV envelope subunits, live recombinant vaccinia virus expressing HIV envelope, and synthetic peptides or virus-like particles derived from yeast, but expressing HIV core proteins, have been used for vaccines that have proved safe . However, large quantities of the subunit HIV antigens are required, and the humoral immune responses are short-lived . Trials with the live recombinant vaccinia virus expressing HIV envelope represent the first use of live recombinant virus in humans . Further studies are under way in France with an avian poxvirus vector, which is host restricted and therefore potentially safer . STudies with combinations of live recombinant virus vaccines followed by immunization with purified subunits indicate that this approach may stimulate both cellular immunity and neutralizing antibodies at higher concentrations than previously observed . The WHO has identified sites in Brazil, Rwanda, Thailand, and Uganda for large scale trials of the efficacy of AIDS vaccines that will probably begin within 5 years . J Mol Biol, 1993 Apr 5, 230(3), 787 - 99 Structure of the sequences adjacent to the centromeric alphoid satellite DNA array on the human Y chromosome; Cooper KF et al.; Eighteen yeast artificial chromosome (YAC) clones containing alphoid satellite DNA and adjacent sequences from the human Y chromosome have been identified from three different YAC libraries . Restriction site mapping of the genomic alphoid arrays and the YACs has allowed seven of the alphoid clones to be positioned on the arrays . Three clones extend into flanking sequences . At one edge the alphoid DNA is highly diverged and is flanked by a small block of the 48 base-pair satellite, dispersed moderately repeated sequences and a separate short alphoid array . More distal sequences are Y-specific . At the other edge there is much less divergence and the alphoid DNA is flanked by an Alu sequence and the five base-pair satellite. Science, 1993 Apr 2, 260(5104), 58 - 63 DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor; Schaeffer L et al.; The human BTF2 basic transcription factor (also called TFIIH), which is similar to the delta factor in rat and factor b in yeast, is required for class II gene transcription . A strand displacement assay was used to show that highly purified preparation of BTF2 had an adenosine triphosphate-dependent DNA helicase activity, in addition to the previously characterized carboxyl-terminal domain kinase activity . Amino acid sequence analysis of the tryptic digest generated from the 89-kilodalton subunit of BTF2 indicated that this polypeptide corresponded to the ERCC-3 gene product, a presumed helicase implicated in the human DNA excision repair disorders xeroderma pigmentosum and Cockayne's syndrome . These findings suggest that transcription and nucleotide excision repair may share common factors and hence may be considered to be functionally related. Yeast, 1993 Apr, 9(4), 331 - 8 Peroxisomal amine oxidase of Hansenula polymorpha does not require its SRL-containing C-terminal sequence for targeting; Faber KN et al.; Amine oxidase (AMO) is a peroxisomal matrix protein of Hansenula polymorpha, which is induced during growth of the yeast in media containing primary amines as a sole nitrogen source . The deduced amino acid sequence of the protein contains an SRL sequence at nine amino acids from the C-terminus . In this study, we have examined the possible role of the SRL motif in sorting of AMO to peroxisomes by mutating the corresponding gene sequence . For this purpose, we have developed a DNA construct that is specifically integrated into the AMO locus of the H . polymorpha genome, placing the mutant gene under the control of the endogenous AMO promoter and eliminating expression of the wild-type gene . Analysis of a stable transformant, containing the desired gene configuration, showed that mutation of the C-terminal sequence neither interfered with correct targeting of the protein into the peroxisome nor displayed significant effects on its activity . From this, it was concluded that the SRL-containing C-terminus is not essential for peroxisomal targeting of AMO in H . polymorpha. Curr Opin Cell Biol, 1993 Apr, 5(2), 214 - 8 Doing the right thing: feedback control and p53; Prives C; Recent evidence suggests that exposure of cells to DNA-damaging agents causes a rise in the levels of the p53 tumor suppressor protein and arrest of progression through the cell cycle . p53 may therefore resemble a member of the RAD gene class identified in yeast, RAD9, which allows cells to repair DNA before continuation of the cell cycle . The evidence that p53 is a sequence-specific, DNA-binding protein that can regulate transcription suggests several ways in which p53 might effect this growth cessation. Curr Opin Cell Biol, 1993 Apr, 5(2), 201 - 6 Turning DNA replication on and off; Roberts JM; DNA replication is coupled to cell cycle progression at a major regulatory point in the G1 phase of the cell cycle . At this point, the catalytic subunit of a protein kinase (encoded by the CDC28 gene in budding yeast or the homologous CDC2 gene in other eukaryotes) is activated by binding to a positively acting regulatory subunit, a cyclin . Recent research has revealed evidence for two pathways that might connect these kinases to the proteins that replicate DNA: activation of an essential replication factor, or removal of the block that limits genome duplication to once per cell cycle. Electrophoresis, 1993 Apr, 14(4), 349 - 54 Influence of the agarose matrix in pulsed-field electrophoresis; Kirkpatrick FH et al.; Properties of agarose potentially relevant to PFGE (pulsed-field gel electrophoresis) are reviewed, and some new information is presented . Agarose polymers appear to have molecular weights in the range of 100,000 to 200,000 Da, but this is not tightly related to the effective gel strength . Agarose has some residual charge, and hence exhibits electroendosmosis (EEO) . It is possible to markedly increase the speed of separation of DNA molecules by using agarose of low EEO, especially in low ionic strength, non-borate buffers . This increase is especially noticeable in the relatively long experiments required for separation of large DNAs . It is also possible to increase the range of separation in a single run by use of step gradients of agarose concentration, which allows visualization of yeast chromosomes and lambda-phage restriction fragments in the same lane . Because of the strong influence of concentration on separation, it may be useful for investigators to control water content and related variables . Our lack of knowledge of the detailed microstructure of gels may be barrier to complete understanding of PFGE. Biophys J, 1993 Apr, 64(4), 1306 - 22 New physical concepts for cell amoeboid motion; Evans E; Amoeboid motion of cells is an essential mechanism in the function of many biological organisms (e.g., the regiment of scavenger cells in the immune defense system of animals) . This process involves rapid chemical polymerization (with numerous protein constituents) to create a musclelike contractile network that advances the cell over the surface . Significant progress has been made in the biology and biochemistry of motile cells, but the physical dynamics of cell spreading and contraction are not well understood . The reason is that general approaches are formulated from complex mass, momentum, and chemical reaction equations for multiphase-multicomponent flow with the nontrivial difficulty of moving boundaries . However, there are strong clues to the dynamics that allow bold steps to be taken in simplifying the physics of motion . First, amoeboid cells often exhibit exceptional kinematics, i.e., steady advance and retraction of local fixed-shape patterns . Second, recent evidence has shown that cell projections "grow" by polymerization along the advancing boundary of the cell . Together, these characteristics represent a local growth process pinned to the interfacial contour of a contractile network . As such, the moving boundary becomes tractable, but subtle features of the motion lead to specific requirements for the chemical nature of the boundary polymerization process . To demonstrate these features, simple examples for limiting conditions of substrate interaction (i.e., "strong" and "weak" adhesion) are compared with data from experimental studies of yeast particle engulfment by blood granulocytes and actin network dynamics in fishscale keratocytes. Int J Syst Bacteriol, 1993 Apr, 43(2), 329 - 37 Five new Legionella species isolated from water; Dennis PJ et al.; Fourteen Legionella-like strains isolated from aquatic sources have been characterized serologically, biochemically, and in terms of DNA relatedness . The strains grew on buffered charcoal-yeast extract agar but not on blood agar and displayed phenotypic characteristics typical of the family Legionellaceae, including a requirement for cysteine, cellular fatty acid compositions in which branched-chain acids predominate, and the possession of isoprenoid quinones of the ubiquinone series with more than 10 isoprene units in their side chains . All were nonfermentative, lacked urease, were incapable of nitrate reduction, and reacted positively with a DNA probe specific for the Legionellaceae . DNA hybridization studies in which the hydroxyapatite method was used demonstrated that the strains represented five new species of the genus Legionella . Nine of the strains were more than 90% interrelated, and the name Legionella londiniensis sp . nov . is proposed for this group . Two strains formed a second hybridization group, for which the name Legionella nautarum sp . nov . is proposed, while the three remaining species, Legionella geestiana sp . nov., Legionella quateirensis sp . nov., and Legionella worsleiensis sp . nov., are each represented by a single strain . The levels of relatedness of the new species to each other are 23% or less, and the levels of relatedness to other members of the genus ranged from 0 to 36% . L . geestiana, L . nautarum, and L . londiniensis are serologically unrelated to all other known Legionella species . L . worsleiensis cannot be separated from Legionella pneumophila serogroup 4 by serological methods and is also serologically indistinguishable from L . quateirensis; distinctions may be made on the basis of fatty acid composition and biochemical reactions. Genomics, 1993 Apr, 16(1), 169 - 72 Microdissection and microcloning of genomic DNA markers from human chromosomal region 11q23; Seki N et al.; A human genomic DNA library was constructed by using a microdissection-microcloning procedure with polymerase chain reaction (PCR) techniques on DNA from the chromosome 11q23 region . A total of 450 recombinant pUC clones were isolated from the library . Their insert sizes ranged from 150 to 850 bp with a mean of 320 bp . Fifty pUC clones were randomly selected and analyzed in detail . Southern blot analyses showed that 21 (42%) clones were unique DNA sequences, 20 (40%) clones were repetitive sequences, and 9 (18%) clones had no detectable hybridization . The unique sequences were used further in a secondary screening of a partially digested human genomic DNA library constructed in phage vector, and 4 clones were isolated . The chromosomal locations of these phage clones were confirmed to be in the q23 region of chromosome 11 by fluorescence in situ suppression hybridization . These pUC microclones isolated from the chromosomal region-specific genomic DNA library will be useful in the construction of physical contig maps with yeast artificial chromosome and/or cosmid clones and in the positional cloning of disease-associated genes localized to the q23 region of chromosome 11. Plant Cell, 1993 Apr, 5(4), 433 - 42 Protein isoprenylation in suspension-cultured tobacco cells; Randall SK et al.; Many mammalian and yeast proteins, including small ras-like GTP binding proteins, heterotrimeric G protein gamma subunits, and nuclear lamins, have been shown to be covalently linked to isoprenoid derivatives of mevalonic acid . Isoprenylation of these proteins is required for their assembly into membranes and, hence, for their biological activity . In this report, it is shown that cultured tobacco cells, when pretreated with an inhibitor of endogenous mevalonic acid synthesis (lovastatin), incorporate radioactivity from 14C-mevalonic acid into proteins . Most of these proteins are membrane associated, and many are similar in mass to mammalian ras-like GTP binding proteins and nuclear lamins . Furthermore, it is shown that tobacco cell extracts catalyze the transfer of radioactivity from 3H-farnesyl pyrophosphate and 3H-geranylgeranyl pyrophosphate to protein substrates in vitro . These studies indicate the presence of at least two distinct prenyl:protein transferases in tobacco extracts: one that utilizes farnesyl pyrophosphate and preferentially modifies a substrate protein with a CAIM carboxy terminus (farnesyl:protein transferase) and one that utilizes geranylgeranyl pyrophosphate and preferentially modifies a substrate protein with a CAIL carboxy terminus (geranylgeranyl:protein transferase type I) . This work provides a basis for future work on the role of protein isoprenylation in plant cell growth, signal transduction, and membrane biogenesis. J Leukoc Biol, 1993 Apr, 53(4), 427 - 38 Transmembrane signaling pathways involved in phagocytosis and associated activation of NADPH oxidase mediated by Fc gamma Rs in human neutrophils; Della Bianca V et al.; We have previously shown that in neutrophils classical transmembrane signaling consisting of increased {Ca2+}i and hydrolysis of phospholipids was not essential for phagocytosis mediated by more than one receptor (yeast-IgG, yeast-C3b/bi, yeast-Con A) . This work deals with the role of this transmembrane signaling in phagocytosis of erythrocyte (E) IgG, which is mediated only by receptors for IgG (Fc gamma Rs) . The ingestion of E-IgG was associated with an increase in {Ca2+}i and production of inositol phosphates, phosphatidic acid, diacylglycerol, and arachidonic acid, via activation of phospholipases C, D and A2 . Related to the same number of particles ingested, the respiratory burst and the transmembrane signaling during phagocytosis of E-IgG were much smaller than during phagocytosis of yeast-IgG . In Ca(2+)-depleted neutrophils, where the increase in {Ca2+}i and hydrolysis of phospholipids were lacking, the phagocytosis of E-IgG was depressed by about 60%; the respiratory burst was also depressed due to the decrease of ingestion and of stimulation of NADPH oxidase by residual phagocytosis . Pertussis toxin (PT) did not inhibit the phagocytosis of E-IgG but depressed by about 40% the stimulation of lipidic transmembrane signaling and the respiratory burst in normal neutrophils . In Ca(2+)-depleted neutrophils the toxin was without effect on ingestion and respiratory burst . Staurosporine did not inhibit the ingestion of E-IgG in normal and Ca(2+)-depleted neutrophils but depressed by 30-40% the respiratory burst in normal and not in Ca(2+)-depleted neutrophils . Genistein, an inhibitor of tyrosine kinase, did not inhibit the ingestion of E-IgG but depressed by 30-40% the respiratory burst both in normal and Ca(2+)-depleted neutrophils . These results demonstrate the following findings in human neutrophils . (1) Contrary to the phagocytosis mediated by more than one receptor (yeast-IgG, yeast-Con A, yeast-C3b/bi), the transmembrane signaling involving increase in {Ca2+}i and hydrolysis of phospholipids plays a role in the phagocytosis and respiratory burst mediated by Fc gamma Rs alone . Thus, different signal transduction pathways can be involved in phagocytosis and associated respiratory burst depending on the receptor or combination of receptors activated . (2) Fc gamma Rs alone promote phagocytosis with two signaling pathways independent of and dependent on {Ca2+}i changes and phospholipid hydrolysis and insensitive to PT, staurosporine, and genistein . (3) The signaling pathways promoting phagocytosis triggered by Fc gamma Rs alone are in some way, or at some step, different from those that activate the respiratory burst. Hum Genet, 1993 Apr, 91(3), 199 - 204 Development of a sequence-tagged site for the centromere of chromosome 10: its use in cytogenetic and physical mapping; Howe JR et al.; We sequenced the alphoid centromere probe p alpha 10RP8 (D10Z1), aligned it to three published consensus sequences, and developed a sequence-tagged site (STS), sJRH-2, based upon oligonucleotide primers having two 3' mismatches with these consensus sequences . Polymerase chain reaction (PCR) amplification using genomic DNA from a somatic cell hybrid panel representing all human chromosomes demonstrated amplification from only those cell lines containing chro