Showing papers on "Gene published in 1995"

Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes.

Abstract: We constructed nine sets of oligonucleotide primers on the basis of the results of DNA hybridization of cloned genes from Neurospora crassa and Aspergillus nidulans to the genomes of select filamentous ascomycetes and deuteromycetes (with filamentous ascomycete affiliations). Nine sets of primers were designed to amplify segments of DNA that span one or more introns in conserved genes. PCR DNA amplification with the nine primer sets with genomic DNA from ascomycetes, deuteromycetes, basidiomycetes, and plants revealed that five of the primer sets amplified a product only from DNA of the filamentous ascomycetes and deuteromycetes. The five primer sets were constructed from the N. crassa genes for histone 3, histone 4, beta-tubulin, and the plasma membrane ATPase. With these five primer sets, polymorphisms were observed in both the size of and restriction enzyme sites in the amplified products from the filamentous ascomycetes. The primer sets described here may provide useful tools for phylogenetic studies and genome analyses in filamentous ascomycetes and deuteromycetes (with ascomycete affiliations), as well as for the rapid differentiation of fungal species by PCR.

Transcriptional activation by tetracyclines in mammalian cells

Abstract: A transcriptional transactivator was developed that fuses the VP16 activation domain with a mutant Tet repressor from Escherichia coli. This transactivator requires certain tetracycline (Tc) derivatives for specific DNA binding. Thus, addition of doxycycline to HeLa cells that constitutively synthesized the transactivator and that contained an appropriate, stably integrated reporter unit rapidly induced gene expression more than a thousandfold. The specificity of the Tet repressor-operator-effector interaction and the pharmacological characteristics of Tc's make this regulatory system well suited for the control of gene activities in vivo, such as in transgenic animals and possibly in gene therapy.

Candidate gene for the chromosome 1 familial Alzheimer's disease locus

Abstract: A candidate gene for the chromosome 1 Alzheimer's disease (AD) locus was identified (STM2). The predicted amino acid sequence for STM2 is homologous to that of the recently cloned chromosome 14 AD gene (S182). A point mutation in STM2, resulting in the substitution of an isoleucine for an asparagine (N141l), was identified in affected people from Volga German AD kindreds. This N141l mutation occurs at an amino acid residue that is conserved in human S182 and in the mouse S182 homolog. The presence of missense mutations in AD subjects in two highly similar genes strongly supports the hypothesis that mutations in both are pathogenic.

The minimal gene complement of Mycoplasma genitalium

Abstract: The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.

Familial Alzheimer's disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer's disease type 3 gene

Abstract: We report the cloning of a novel gene (E5-1) encoded on chromosome 1 which has substantial nucleotide and amino-acid sequence similarity to the S182 gene on chromosome 14q24.3. Mutations, including three new missense mutations in the S182 gene, are associated with the AD3 subtype of early-onset familial Alzheimer's disease (AD). Both the E5-1 and the S182 proteins are predicted to be integral membrane proteins with seven membrane-spanning domains, and a large exposed loop between the sixth and seventh transmembrane domains. Analysis of the nucleotide sequence of the open reading frame (ORF) of the E5-1 gene led to the discovery of two missense substitutions at conserved amino-acid residues in affected members of pedigrees with a form of familial AD that has a later age of onset than the AD3 subtype (50-70 years versus 30-60 years for AD3). These observations imply that the E5-1 gene on chromosome 1 and the S182 gene on chromosome 14q24.3 are members of a family of genes (presenilins) with related functions, and indicates that mutations in conserved residues of E5-1 could also play a role in the genesis of AD. Our results also indicate that still other AD susceptibility genes exist.

A Receptor Kinase-Like Protein Encoded by the Rice Disease Resistance Gene, Xa21

Abstract: The rice Xa21 gene, which confers resistance to Xanthomonas oryzae pv. oryzae race 6, was isolated by positional cloning. Fifty transgenic rice plants carrying the cloned Xa21 gene display high levels of resistance to the pathogen. The sequence of the predicted protein, which carries both a leucine-rich repeat motif and a serine-threonine kinase-like domain, suggests a role in cell surface recognition of a pathogen ligand and subsequent activation of an intracellular defense response. Characterization of Xa21 should facilitate understanding of plant disease resistance and lead to engineered resistance in rice.

A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. - eScholarship

Abstract: The rice Xa21 gene, which confers resistance to Xanthomonas oryzae pv. oryzae race 6, was isolated by positional cloning. Fifty transgenic rice plants carrying the cloned Xa21 gene display high levels of resistance to the pathogen. The sequence of the predicted protein, which carries both a leucine-rich repeat motif and a serine-threonine kinase-like domain, suggests a role in cell surface recognition of a pathogen ligand and subsequent activation of an intracellular defense response. Characterization of Xa21 should facilitate understanding of plant disease resistance and lead to engineered resistance in rice.

Inducible gene targeting in mice

Abstract: A method of gene targeting that allows the inducible inactivation of a target gene in mice is presented The method uses an interferon-responsive promoter to control the expression of Cre recombinase Here, Cre was used to delete a segment of the DNA polymerase beta gene flanked by IoxP recombinase recognition sites Deletion was complete in liver and nearly complete in lymphocytes within a few days, whereas partial deletion was obtained in other tissues This method can be used for the inducible inactivation of any other gene in vivo

Somatic Generation of Antibody Diversity1

Abstract: Publisher Summary This chapter reviews the somatic generation of antibody diversity. It was shown that a typical antibody molecule is composed of two identical light chains and two identical heavy chains. It had also been found that each of these two types of chain exhibits great sequence variability in the amino terminal region between one antibody molecule and the next and no sequence variability in the carboxyl terminal regions. Use of restriction enzymes and recombinant DNA methods allowed resolution of a long-standing and central issue in immunology, the genetic origins of antibody diversity. It turned out that an organism does not inherit even a single complete gene for antibody polypeptide chains. Rather, the genetic information is transmitted in germline as no more than several hundred gene segments. Through a series of specialized somatic recombination occurring specifically during the differentiation of B lymphocytes, these gene segments are assembled into tens of thousands of complete genes. In the immune system, organisms have exploited two major processes for modification of DNA, recombination and mutation, as a means to diversify somatically the limited amount of inherited genetic information to cope with the vastly diverse antigen universe. Somatic diversification allows the individual organism to generate a virtually limitless number of lymphocyte variants.

Plant Molecular Biology Manual

Abstract: Plant Molecular Biology Manual (Second Edition) is an entirely new manual containing both fundamental and recently described techniques in the area of plant molecular biology. Designed for use in the research laboratory, the Plant Molecular Biology Manual presents detailed techniques in the areas of plant transformation, recombinant DNA and other nucleic acid manipulations, nuclear run-on and in vitro transcription systems, in situ hybridization and immunodetection systems, protein-nucleic acid interaction analyses, subcellular targeting of proteins in the plant cell, and gene tagging using T-DNA and transposons. This second edition contains more than 40 newly written chapters, including descriptions of subjects such as virus-mediated gene transfer, specialized Agrobacterium strains and T-DNA vectors, nuclear run-on and in vitro transcription systems, non-radioactive detection systems, characterization of transcription factors, nuclear protein targeting, and T-DNA and transposon mutagenesis, not previously described in the first edition.

Transfer of Genes to Humans: Early Lessons and Obstacles to Success

Abstract: Enough information has been gained from clinical trials to allow the conclusion that human gene transfer is feasible, can evoke biologic responses that are relevant to human disease, and can provide important insights into human biology. Adverse events have been uncommon and have been related to the gene delivery strategies, not to the genetic material being transferred. Human gene transfer still faces significant hurdles before it becomes an established therapeutic strategy. However, its accomplishments to date are impressive, and the logic of the potential usefulness of this clinical paradigm continues to be compelling.

Genetics and Biochemistry of Ant hocyanin Biosynthesis

Abstract: Flavonoids represent a large class of secondary plant metabolites, of which anthocyanins are the most conspicuous class, dueto the wide range of colors resulting from their synthesis. Anthocyanins are important to many diverse functions within plants. Synthesis of anthocyanins in petals is undoubtedly intended to attract pollinators, whereas anthocyanin synthesis in seeds and fruits may aid in seed dispersal. Anthocyanins and other flavonoids can also be important as feeding deterrents and as protection against damage from UV irradiation. The existence of such a diverse range of functions and types of anthocyanins raises questions about how these compounds are synthesized and how their synthesis is regulated. The study of the genetics of anthocyanin synthesis began last century with Mendel’s work on flower color in peas. Since that time, there have been periods of intensive study into the genetics and biochemistry of pigment production in a number of different species. In the early studies, genetic loci were correlated with easily observable color changes. After the structures of anthocyanins and other flavonoids were determined, it was possible to correlate single genes with particular structural alterations of anthocyanins or with the presence or absence of particular flavonoids. Mutations in anthocyanin genes have been studied for many years because they are easily identified and because they generally have no deleterious effect on plant growth and development. In most cases, mutations affecting different steps of the anthocyanin biosynthesis pathway were isolated and characterized well before their function was identified or the corresponding gene was isolated. More recently, many genes involved in the biosynthesis of anthocyanin pigments have been isolated and characterized using recombinant DNA technologies. Three species have been particularly important for elucidating the anthocyanin biosynthetic pathway and for isolating genes controlling the biosynthesis of flavonoids: maize (Zea mays), snapdragon (Anfirrhinum majus), and petunia (Wtunia hybrida). Petunia has more recently become the organism of choice for isolating flavonoid biosynthetic genes and studying their interactions and regulation. At least 35 genes are known to affect flower color in petuniawiering and de Vlaming, 1984). Because this field of research has been reviewed fairly extensively in the past (Dooner et al., 1991; van Tunen and MOI, 1991; Gerats and Martin, 1992), in this review we concentrate on the more recent developments in gene isolation and characterization. A review of the genetics of flavonoid biosynthesis in other species was recently covered by Forkmann (1993). The characterization of genetically defined mutations has enabled the order of many reactions in anthocyanin synthesis and their modification to be elucidated. Some reactions have been postulated only on the basis of genetic studies and have not yet been demonstrated in vitro. Chemico-genetic studies have been very important in determining the enzymatic steps involved in anthocyanin biosynthesis and modification. The generation of transposon-tagged mutations and the subsequent cloning of the transposons provided a relatively straightforward means of isolating many genes from maize (Wienand et al., 1990) and snapdragon (Martin et al., 1991). However, a number of genes in the pathway have not been amenable to transposon tagging. Anthocyanin biosynthetic genes have been isolated using a range of methodologies, including protein purification, transposon tagging, differential screening, and polymerase chain reaction (PCR) amplification. Functions of isolated anthocyanin genes can be confirmed by restriction fragment length polymorphism (RFLP) mapping, complementation, or expression in heterologous systems. Reverse genetics has also been used recently to identify gene function; this requires a welldefined pathway to correlate phenotype with gene function. Once a gene has been isolated from one species, it is usually a straightforward task to isolate the homologous gene from other species by using the original clone as a molecular probe.

Simultaneous identification of bacterial virulence genes by negative selection

Abstract: An insertional mutagenesis system that uses transposons carrying unique DNA sequence tags was developed for the isolation of bacterial virulence genes. The tags from a mixed population of bacterial mutants representing the inoculum and bacteria recovered from infected hosts were detected by amplification, radiolabeling, and hybridization analysis. When applied to a murine model of typhoid fever caused by Salmonella typhimurium, mutants with attenuated virulence were revealed by use of tags that were present in the inoculum but not in bacteria recovered from infected mice. This approach resulted in the identification of new virulence genes, some of which are related to, but functionally distinct from, the inv/spa family of S. typhimurium.

Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR.

Abstract: A PCR assay that allows simultaneous detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci (Enterococcus faecium, E. faecalis, E. gallinarum, and E. casseliflavus) was developed. This assay was based on specific amplification of internal fragments of genes encoding D-alanine:D-alanine ligases and related glycopeptide resistance proteins. The specificity of the assay was tested on 5 well-characterized glycopeptide-resistant strains and on 15 susceptible enterococcal type strains. Clinical isolates of enterococci that could not be identified to the species level by conventional methods were identified by the PCR test. This assay offers a specific and rapid alternative to antibiotic susceptibility tests, in particular for detection of low-level vancomycin resistance.

The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo

Abstract: Evidence is accumulating that members of the FGF gene family provide signals that act locally to regulate growth and patterning in vertebrate embryos. In this report, we provide a detailed analysis of the mouse Fgf8 gene. We have mapped the Fgf8 locus to the distal region of mouse chromosome 19, and sequenced the 5' coding region of the gene. Our data identify a new coding exon, and locate multiple splice donor and splice acceptor sites that can be used to produce at least seven transcripts encoding a family of secreted FGF8 proteins with different N termini. From these results, it appears that Fgf8 is structurally the most complex member of the FGF family described to date. In the embryo, many of the regions in which Fgf8 RNA is localized are known to direct outgrowth and patterning, including the apical ectodermal ridge of the limb bud, the primitive streak and tail bud, the surface ectoderm overlying the facial primorida and the midbrain-hindbrain junction, suggesting that FGF8 may be a component of the regulatory signals that emanate from these regions.

Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase.

Abstract: The level of genetic variation of human immunodeficiency virus type 1 (HIV-1), a member of the lentivirus genus of the Retroviridae family, is high relative to that of retroviruses in some other genera. The high error rates of purified HIV-1 reverse transcriptase in cell-free systems suggest an explanation for this high genetic variation. To test whether the in vivo rate of mutation during reverse transcription of HIV-1 is as high as predicted by cell-free studies, and therefore higher than that rates of mutation of retroviruses in other genera, we developed an in vivo assay for detecting forward mutations in HIV-1, using the lacZ alpha peptide gene as a reporter for mutations. This system allows the rates and types of mutations that occur during a single cycle of replication to be studied. We found that the forward mutation rate for HIV-1 was 3.4 x 10(-5) mutations per bp per cycle. Base substitution mutations predominated; G-to-A transition mutations were the most common base substitution. The in vivo mutation rates for HIV-1 are three and seven times higher than those previously reported for two other retroviruses, spleen necrosis virus and bovine leukemia virus, respectively. In contrast, our calculated in vivo mutation rate for HIV-1 is about 20-fold lower than the error rate of purified HIV-1 reverse transcriptase, with the same target sequence. This finding indicates that HIV-1 reverse transcription in vivo is not as error prone as predicted from the fidelity of purified reverse transcriptase in cell-free studies. Our data suggest that the fidelity of purified HIV-1 reverse transcriptase may not accurately reflect the level of genetic variation in a natural infection.

Rubinstein-Taybi syndrome caused by mutations in the transcriptional co-activator CBP

Abstract: THE Rubinstein-Taybi syndrome (RTS) is a well-defined syndrome with facial abnormalities, broad thumbs, broad big toes and mental retardation as the main clinical features1-3. Many patients with RTS have been shown to have breakpoints in, and microdeletions of, chromosome 16pl3.3 (refs 4-8). Here we report that all these breakpoints are restricted to a region that contains the gene for the human CREB binding protein (CBP), a nuclear protein participating as a co-activator in cyclic-A IMP-regulated gene expression9-12. We show that RTS results not only from gross chromosomal rearrangements of chromosome 16p, but also from point mutations in the CBP gene itself. Because the patients are heterozygous for the mutations, we propose that the loss of one functional copy of the CBP gene underlies the developmental abnormalities in RTS and possibly the propensity for malignancy.

Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA–binding protein gene

Abstract: We have detected deletions of portions of the Y chromosome long arm in 12 of 89 men with azoospermia (no sperm in semen). No Y deletions were detected in their male relatives or in 90 other fertile males. The 12 deletions overlap, defining a region likely to contain one or more genes required for spermatogenesis (the Azoospermia Factor, AZF). Deletion of the AZF region is associated with highly variable testicular defects, ranging from complete absence of germ cells to spermatogenic arrest with occasional production of condensed spermatids. We find no evidence of YRRM genes, recently proposed as AZF candidates, in the AZF region. The region contains a single–copy gene, DAZ (Deleted in AZoospermia), which is transcribed in the adult testis and appears to encode an RNA binding protein. The possibility that DAZ is AZF should now be explored.

PCR mapping of integrons reveals several novel combinations of resistance genes.

Abstract: The integron is a new type of mobile element which has evolved by a site-specific recombinational mechanism. Integrons consist of two conserved segments of DNA separated by a variable region containing one or more genes integrated as cassettes. Oligonucleotide probes specific for the conserved segments have revealed that integrons are widespread in recently isolated clinical bacteria. Also, by using oligonucleotide probes for several antibiotic resistance genes, we have found novel combinations of resistance genes in these strains. By using PCR, we have determined the content and order of the resistance genes inserted between the conserved segments in the integrons of these clinical isolates. PCR mapping of integrons can be a useful epidemiological tool to study the evolution of multiresistance plasmids and transposons and dissemination of antibiotic resistance genes.

Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence.

Abstract: In an effort to identify new genes and analyse their expression patterns, 174,472 partial complementary DNA sequences (expressed sequence tags (ESTs)), totalling more than 52 million nucleotides of human DNA sequence, have been generated from 300 cDNA libraries constructed from 37 distinct organs and tissues. These ESTs have been combined with an additional 118,406 ESTs from the database dbEST, for a total of 83 million nucleotides, and treated as a shotgun sequence assembly project. The assembly process yielded 29,599 distinct tentative human consensus (THC) sequences and 58,384 non-overlapping ESTs. Of these 87,983 distinct sequences, 10,214 further characterize previously known genes based on statistically significant similarity to sequences in the available databases; the remainder identify previously unknown genes. Thirty tissues were sampled by over 1,000 ESTs each; only eight genes were matched by ESTs from all 30 tissues, and 227 genes were represented in 20 or more of the tissues sampled with more than 1,000 ESTs. Approximately 40% of identified human genes appear to be associated with basic energy metabolism, cell structure, homeostasis and cell division, 22% with RNA and protein synthesis and processing, and 12% with cell signalling and communication.

Structure of the Arabidopsis RPM1 gene enabling dual specificity disease resistance

Abstract: Plants can recognize pathogens through the action of disease resistance (R) genes, which confer resistance to pathogens expressing unique corresponding avirulence (avr) genes. The molecular basis of this gene-for-gene specificity is unknown. The Arabidopsis thaliana RPM1 gene enables dual specificity to pathogens expressing either of two unrelated Pseudomonas syringae avr genes. Despite this function, RPM1 encodes a protein sharing molecular features with recently described single-specificity R genes. Surprisingly, RPM1 is lacking from naturally occurring, disease-susceptible Arabidopsis accessions.

An evaluation of genetic distances for use with microsatellite loci.

Abstract: Mutations of alleles at microsatellite loci tend to result in alleles with repeat scores similar to those of the alleles from which they were derived. Therefore the difference in repeat score between alleles carries information about the amount of time that has passed since they shared a common ancestral allele. This information is ignored by genetic distances based on the infinite alleles model. Here we develop a genetic distance based on the stepwise mutation model that includes allelic repeat score. We adapt earlier treatments of the stepwise mutation model to show analytically that the expectation of this distance is a linear function of time. We then use computer simulations to evaluate the overall reliability of this distance and to compare it with allele sharing and Nei's distance. We find that no distance is uniformly superior for all purposes, but that for phylogenetic reconstruction of taxa that are sufficiently diverged, our new distance is preferable.