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Biomedical subjects

L Hood

Publications and source records attributed to L Hood.

At least 91 records · Page 5Linked to original sources
PubMed1998

Snapshot of a large dynamic replicon in a halophilic archaeon: megaplasmid or minichromosome?

Extremely halophilic archaea, which flourish in hypersaline environments, are known to contain a variety of large dynamic replicons. Previously, the analysis of one such replicon, pNRC100, in Halobacterium sp. strain NRC-1, showed that it undergoes high-frequency insertion sequence (IS) element-mediated insertions and deletions, as well as inversions via recombination between 39-kb-long inverted repeats (IRs). Now, the complete sequencing of pNRC100, a 191,346-bp circle, has shown the presence of 27 IS elements representing eight families. A total of 176 ORFs or likely genes of 850-bp average size were found, 39 of which were repeated within the large IRs. More than one-half of the ORFs are likely to represent novel genes that have no known homologs in the databases. Among ORFs with previously characterized homologs, three different copies of putative plasmid replication and four copies of partitioning genes were found, suggesting that pNRC100 evolved from IS element-mediated fusions of several smaller plasmids. Consistent with this idea, putative genes typically found on plasmids, including those encoding a restriction-modification system and arsenic resistance, as well as buoyant gas-filled vesicles and a two-component regulatory system, were found on pNRC100. However, additional putative genes not expected on an extrachromosomal element, such as those encoding an electron transport chain cytochrome d oxidase, DNA nucleotide synthesis enzymes thioredoxin and thioredoxin reductase, and eukaryotic-like TATA-binding protein transcription factors and a chromosomal replication initiator protein were also found. A multi-step IS element-mediated process is proposed to account for the acquisition of these chromosomal genes. The finding of essential genes on pNRC100 and its property of resistance to curing suggest that this replicon may be evolving into a new chromosome.

Chromosome Mapping↗
PubMed1998

Evolution of a simian immunodeficiency virus pathogen.

Analysis of disease induction by simian immunodeficiency viruses (SIV) in macaques was initially hampered by a lack of molecularly defined pathogenic strains. The first molecularly cloned SIV strains inoculated into macaques, SIVmacBK28 and SIVmacBK44 (hereafter designated BK28 and BK44, respectively), were cases in point, since they failed to induce disease within 1 year postinoculation in any inoculated animal. Here we report the natural history of infection with BK28 and BK44 in inoculated rhesus macaques and efforts to increase the pathogenicity of BK28 through genetic manipulation and in vivo passage. BK44 infection resulted in no disease in four animals infected for more than 7 years, whereas BK28 induced disease in less than half of animals monitored for up to 7 years. Elongation of the BK28 transmembrane protein (TM) coding sequence truncated by prior passage in human cells marginally increased pathogenicity, with two of four animals dying in the third year and one dying in the seventh year of infection. Modification of the BK28 long terminal repeat to include four consensus nuclear factor SP1 and two consensus NF-kappaB binding sites enhanced early virus replication without augmenting pathogenicity. In contrast, in vivo passage of BK28 from the first animal to die from immunodeficiency disease (1.5 years after infection) resulted in a consistently pathogenic strain and a 50% survival time of about 1.3 years, thus corresponding to one of the most pathogenic SIV strains identified to date. To determine whether the diverse viral quasispecies that evolved during in vivo passage was required for pathogenicity or whether a more virulent virus variant had evolved, we generated a molecular clone composed of the 3' half of the viral genome derived from the in vivo-passaged virus (H824) fused with the 5' half of the BK28 genome. Kinetics of disease induction with this cloned virus (BK28/H824) were similar to those with the in vivo-passaged virus, with four of five animals surviving less than 1.7 years. Thus, evolution of variants with enhanced pathogenicity can account for the increased pathogenicity of this SIV strain. The genetic changes responsible for this virulent transformation included at most 59 point mutations and 3 length-change mutations. The critical mutations were likely to have been multiple and dispersed, including elongation of the TM and Nef coding sequences; changes in RNA splice donor and acceptor sites, TATA box sites, and Sp1 sites; multiple changes in the V2 region of SU, including a consensus neutralization epitope; and five new N-linked glycosylation sites in SU.

Amino Acid Sequence↗
PubMed1997

Gene families: the taxonomy of protein paralogs and chimeras.

Ancient duplications and rearrangements of protein-coding segments have resulted in complex gene family relationships. Duplications can be tandem or dispersed and can involve entire coding regions or modules that correspond to folded protein domains. As a result, gene products may acquire new specificities, altered recognition properties, or modified functions. Extreme proliferation of some families within an organism, perhaps at the expense of other families, may correspond to functional innovations during evolution. The underlying processes are still at work, and the large fraction of human and other genomes consisting of transposable elements may be a manifestation of the evolutionary benefits of genomic flexibility.

Amino Acid Sequence↗
PubMed1997

MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclXL and initiates cell death.

Activation of the cascade of proteolytic caspases has been identified as the final common pathway of apoptosis in diverse biological systems. We have isolated a gene, termed MRIT, that possesses overall sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases. However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus sequence QAC(R/Q)G. Nonetheless MRIT activates caspase-dependent death. Using yeast two-hybrid assays, we demonstrate that MRIT associates with caspases possessing large and small prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD. In addition, MRIT simultaneously and independently interacts with BclXL and FLICE in mammalian cells. Thus, MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family member.

Adult↗
PubMed1997

Cell-cell interaction in prostate gene regulation and cytodifferentiation.

To examine the role of intercellular interaction on cell differentiation and gene expression in human prostate, we separated the two major epithelial cell populations and studied them in isolation and in combination with stromal cells. The epithelial cells were separated by flow cytometry using antibodies against differentially expressed cell-surface markers CD44 and CD57. Basal epithelial cells express CD44, and luminal epithelial cells express CD57. The CD57+ luminal cells are the terminally differentiated secretory cells of the gland that synthesize prostate-specific antigen (PSA). Expression of PSA is regulated by androgen, and PSA mRNA is one of the abundant messages in these cells. We show that PSA expression by the CD57+ cells is abolished after prostate tissue is dispersed by collagenase into single cells. Expression is restored when CD57+ cells are reconstituted with stromal cells. The CD44+ basal cells possess characteristics of stem cells and are the candidate progenitors of luminal cells. Differentiation, as reflected by PSA production, can be detected when CD44+ cells are cocultured with stromal cells. Our studies show that cell-cell interaction plays an important role in prostatic cytodifferentiation and the maintenance of the differentiated state.

Androgens↗
PubMed1997

Low fidelity mutants in the O-helix of Thermus aquaticus DNA polymerase I.

We screened 67 mutants in the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) for altered fidelity of DNA synthesis. These mutants were obtained (Suzuki, M., Baskin, D., Hood, L., and Loeb, L. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9670-9675) by substituting an oligonucleotide containing random sequences for codons 659-671, and selecting for complementation of a growth defect in Escherichia coli caused by temperature-sensitive host pol I. Thirteen mutants decreased fidelity in a screen that employed primer extension reactions lacking one of four complementary deoxynucleoside triphosphates (dNTPs). Three mutants were purified and exhibited 29-68% of wild-type specific activity. Homogeneous polymerases A661E, A661P, and T664R extended primers further than the wild-type, synthesizing past template nucleotides for which the complementary dNTP was absent. The data indicate that both misinsertion of incorrect nucleotides and extension of mispaired primer termini were increased. In a lacZalpha forward mutation assay, A661E and T664R yielded mutation frequencies at least 7- and 25-fold greater, respectively, than that of the wild-type polymerase. These findings emphasize the importance of the O-helix in substrate recognition and are compatible with a role for pyrophosphate release in enhancing fidelity of DNA synthesis.

Base Sequence↗
PubMed1997

Identification of differentially expressed prostate genes: increased expression of transcription factor ETS-2 in prostate cancer.

BACKGROUND: Little is known about the genetic events in the malignant transformation of prostatic cells. This is due in large measure to the cellular heterogeneity of the prostate. METHODS: An amplification method was devised to synthesize cDNA from small samples of cancer and benign tissues of the same resected glands. Differential gene expression of candidate informative markers between cancer and benign was screened by the polymerase chain reaction with gene-specific oligonucleotide primers. RESULTS: The expression of a transcription factor, ETS-2, was shown to be elevated in some cancer specimens. Elevated expression was also noted for neuron-specific enolase (NSE) and another transcription factor, SEF2. CONCLUSIONS: Our method can be used to identify quickly genes that are differentially expressed between benign and cancerous prostate cells. Transcription factors, such as ETS-2, may play a significant role in cancer progression.

Cell Transformation, Neoplastic↗
PubMed1997

Evolution and selection of primate T cell antigen receptor BV8 gene subfamily.

The set of potential T cell receptor specificities is highly diverse. The relative contributions of T cell receptor (TCR) V beta gene segment polymorphisms, duplications, deletions, and gene conversions to this final T cell receptor protein diversity are unknown. To study these mechanisms, we sequenced and compared closely related primate TCR gene segments from BV8S1, S2, and S5. Interspecies comparisons show that these gene segments have sustained multiple duplication, gene conversion, and deletion events during the last 35 million years of anthropoid primate evolution. BV8 coding sequences are generally conserved with respect to their flanking noncoding sequences, but we find no evidence for positive or negative selection in sequences coding for the first two putative complementarity-determining (ligand-binding) regions. Sequences of TCRBV8 gene segments from unrelated humans demonstrate no nonsynonymous substitutions in nonleader regions of either the BV8S1 or S2 gene segments. We conclude that gene duplication, deletion, and conversion mechanism contribute in a substantial way to the overall diversity of the TCRBV8 gene segment repertoire in primate evolution and that germline substitutions and consequent polymorphisms in CDRs 1 and 2 of these gene segments probably do not play an active role in generating TCR beta chain protein variation.

Adult↗
PubMed1997

The molecular evolution of the vertebrate trypsinogens.

We expand the already large number of known trypsinogen nucleotide and amino acid sequences by presenting additional trypsinogen sequences from the tunicate (Boltenia villosa), the lamprey (Petromyzon marinus), the pufferfish (Fugu rubripes), and the frog (Xenopus laevis). The current array of known trypsinogen sequences now spans the entire vertebrate phylogeny. Phylogenetic analysis is made difficult by the presence of multiple isozymes within species and rates of evolution that vary highly between both species and isozymes. We nevertheless present a Fitch-Margoliash phylogeny constructed from pairwise distances. We employ this phylogeny as a vehicle for speculation on the evolution of the trypsinogen gene family as well as the general modes of evolution of multigene families. Unique attributes of the lamprey and tunicate trypsinogens are noted.

Amino Acid Sequence↗
PubMed1997

A highly conserved apoptotic suppressor gene is located near the chicken T-cell receptor alpha chain constant region.

A cosmid clone containing the chicken T-cell receptor alpha chain constant region (TCRAC) was sequenced. The cosmid contains the TCRAC gene, six putative joining gene segments (TCRAJ), and surprisingly, a chicken homologue for the human apoptotic suppressor gene, defender against cell death (DAD1). The DAD1 gene is 6.3 kilobases downstream of the TCRAC gene and has an inverted transcription orientation with respect to the TCRAC gene. The cDNA for the chicken DAD1 gene is 597 base pairs in length and encodes a highly conserved hydrophobic protein. The proximal location of DAD1 to the TCRAC locus has also been confirmed in both humans and mouse. The location of the DAD1 gene suggests that DAD1 may play an important role in T-cell related apoptotic activities.

Amino Acid Sequence↗
PubMed1997

TCRV and TCRJ gene usage in MBP responding T cells from (B10.PL x PL/J)F1 mice is biased towards that of B10.PL mice.

We analyzed myelin basic protein (MBP) specific T cell hybridoma clones from (B10.PL x PL/J)F1 mice. MBP-reacting T cell hybridomas from F1 mice preferentially expressed B10.PL TcraV2.3 (53%) and B10.PL TcraV4.2 (13%) with minor expression of TcraV4.4 (13%) gene segments. A dominant expression of TcrbV8.2 (73%) accompanying with TcrbV8.1 (20%) and TcrbV13 (7%) gene segments have been identified in these MBP-reacting T cell hybridomas from F1 mice. There was less restrictive but non-random usage of the TcraJ and TcrbJ gene segments. Overall, the MBP-reacting T cell hybridomas from (B10.PL x PL/J)F1 mice were dominated by the MBP-reacting T cell pattern seen in B10.PL mice.

Amino Acid Sequence↗
PubMed1997

Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-kappaB pathway.

Death receptor 4 (DR4) is a recently described receptor for the cytotoxic ligand TRAIL that reportedly uses a FADD-independent pathway to induce apoptosis and does not activate the NF-kappaB pathway. We have isolated a new member of the tumor necrosis factor receptor (TNFR) family, designated DR5, which bears a high degree of sequence homology to DR4. However, contrary to the previous reports, both DR4- and DR5-induced apoptosis can be blocked by dominant-negative FADD, and both receptors can activate NF-kappaB using a TRADD-dependent pathway. Finally, both receptors can interact with FADD, TRADD, and RIP. Thus, both DR5 and DR4 use FADD, TRADD, and RIP in their signal transduction pathways, and FADD is the common mediator of apoptosis by all known death domain-containing receptors.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1.

Alagille syndrome is an autosomal dominant disorder characterized by abnormal development of liver, heart, skeleton, eye, face and, less frequently, kidney. Analyses of many patients with cytogenetic deletions or rearrangements have mapped the gene to chromosome 20p12, although deletions are found in a relatively small proportion of patients (< 7%). We have mapped the human Jagged1 gene (JAG1), encoding a ligand for the developmentally important Notch transmembrane receptor, to the Alagille syndrome critical region within 20p12. The Notch intercellular signalling pathway has been shown to mediate cell fate decisions during development in invertebrates and vertebrates. We demonstrate four distinct coding mutations in JAG1 from four Alagille syndrome families, providing evidence that it is the causal gene for Alagille syndrome. All four mutations lie within conserved regions of the gene and cause translational frameshifts, resulting in gross alterations of the protein product Patients with cytogenetically detectable deletions including JAG1 have Alagille syndrome, supporting the hypothesis that haploinsufficiency for this gene is one of the mechanisms causing the Alagille syndrome phenotype.

Alagille Syndrome↗
PubMed1997

Linkage analysis of 49 high-risk families does not support a common familial prostate cancer-susceptibility gene at 1q24-25.

Linkage of a putative prostate cancer-susceptibility locus (HPC1) to chromosome 1q24-25 has recently been reported. Confirmation of this linkage in independent data sets is essential because of the complex nature of this disease. Here we report the results of a linkage analysis using 10 polymorphic markers spanning approximately 37 cM in the region of the putative HPC1 locus in 49 high-risk prostate cancer families. Data were analyzed by use of two parametric models and a nonparametric method. For the parametric LOD-score method, the first model was identical to the original report by Smith and coworkers ("Hopkins"), and the second was based on a segregation analysis previously reported by Carter and coworkers ("Seattle"). In both cases, our results do not confirm the linkage reported for this region. Calculated LOD scores from the two-point analysis for each marker were highly negative at small recombination fractions. Multipoint LOD scores for this linkage group were also highly negative. Additionally, we were unable to demonstrate heterogeneity within the data set, using HOMOG. Although these data do not formally exclude linkage of a prostate cancer-susceptibility locus at HPC1, it is likely that other prostate cancer-susceptibility loci play a more critical role in the families that we studied.

Adult↗
PubMed1997

Hopper: software for automating data tracking and flow in DNA sequencing.

MOTIVATION: Genome-scale DNA sequencing is a multistep process in which large numbers of small template clones are propagated, purified, sequenced and analyzed on acrylamide gels. A significant challenge to these projects is the scale at which the data handling must be done. Hence, large-scale sequencing facilities will benefit from tracking template DNA information (purification methods, reaction and electrophoresis conditions) in a systematic fashion. A lack of software tools that support automated sample entry, and automatic data storage, retrieval and analysis are a major hindrance to recording and using laboratory workflow information to monitor the overall quality of data production. RESULTS: The UNIX file system has been used to prototype automation of the flow of data from the ABI sequencer to a data repository. Data are automatically processed by a central Perl program, Hopper, which runs a series of programs that analyze data quality (read length estimate, fraction of indeterminate bases, and number of contaminating and repetitive sequences), assemble shotgun sequence data, and generates simple reports describing the results.

Database Management Systems↗
PubMed1997

Analysis of the 1.1-Mb human alpha/delta T-cell receptor locus with bacterial artificial chromosome clones.

Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents. The 1.1-Mb alpha/delta T-cell receptor locus of humans was mapped and partially sequenced with BAC clones. Seventeen BAC clones covered the 1.1-Mb alpha/delta locus, with the exception of one small gap that was expected from the coverage that a 3.7-fold BAC library is likely to provide. The end sequences of the BAC inserts could be obtained directly from the BAC DNA by sequencing with the chain terminator chemistry. Five complete BAC inserts were sequenced directly by the shotgun approach. The ends of the 17 BAC inserts were distributed evenly across the locus. By several independent criteria, the BAC clones faithfully represented the genomic DNA, with the exception of a single clone with a 68-kb deletion. These BAC features led to the proposal of a new approach to sequence the human genome.

Bacteriophage M13↗