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At least 307 records · Page 17Linked to original sources
PubMed1987

Physical mapping of genes and sequences at the end of the human X chromosome short arm.

Human-rodent somatic cell hybrids containing deleted and translocated human X chromosomes have been used to map genes and sequences in and around the pseudoautosomal region. The following order was found: (DXS69, DXS70, DXS143)-(DXS31, STS)-MIC2. This order is consistent with the known inheritance patterns of DXS31, STS and MIC2. Assuming that the translocations and deletions we have studied are not complex rearrangements, we conclude that the pseudoautosomal region consists of less than 5 X 10(6) bp of DNA.

Animals↗
PubMed1995

Peroxisomal membrane protein PMP68 of mouse liver: cloning of a cDNA encompassing the nucleotide binding fold and epitope mapping of monoclonal antibodies to the expressed protein.

We have isolated and sequenced a cDNA which encodes 376 amino acids toward the carboxy-terminus, and encompassing the putative nucleotide binding fold, of PMP68 (mouse liver peroxisomal integral membrane protein of 68 kDa) the major integral membrane protein of mouse liver peroxisomes. The protein sequence predicted from this cDNA shows 97.9% amino acid identity to this same region of rat liver PMP70, a member of the ATP-binding cassette protein superfamily (K. Kamijo, S. Taketani, S. Yokota, T. Osumi, and T. Hashimoto, 1990, J. Biol. Chem. 265, 4534-4540). The section of the cDNA encoding the hydrophilic and putative cytoplasmic domain of PMP68 was expressed as a recombinant fusion protein in bacteria. Two monoclonal antibodies raised against this protein have been epitope-mapped to peptides generated by cyanogen bromide cleavage of the fusion protein. Antibody 1A4 recognizes a peptide whose sequence contains the first motif of the putative nucleotide binding fold of PMP68, and antibody 8F11 recognizes a carboxy-terminal peptide which includes the second motif of this nucleotide binding fold. These antibodies are expected to be useful in the elucidation of the biological function of this putative membrane transporter.

ATP-Binding Cassette Transporters↗
PubMed1979

Genetic mapping of a mutation affecting pyridine nucleotide transhydrogenase in Escherichia coli.

A mutation, pnt-1, causing loss of pyridine nucleotide transhydrogenase activity in Escherichia coli, was mapped by assaying for the enzyme in extracts of recombinant strains produced by conjugation, F-duction, and P1 transduction. The site of this mutation was near min 35, counterclockwise from man, and it co-transduced 59% with man. The mutation was associated with loss from the cell membrane fraction of energy-independent and adenosine 5'-triphosphate-dependent transhydrogenase activities, but reduced nicotinamide adenine dinucleotide dehydrogenase activity was not affected. Strains were constructed which lack phosphoglucoisomerase (pgi-2) and which carry either pnt+ or pnt-1. Although such strains, when grown on glucose, are expected to produce a large excess of reduced nicotinamide adenine dinucleotide phosphate, the growth rate was not affected by the pnt-1 allele.

Cell Membrane↗
PubMed1997

Evidence for a switch in the mode of human papillomavirus type 16 DNA replication during the viral life cycle.

The study of human papillomavirus type 16 (HPV-16) replication has been impaired because of the lack of a cell culture system that stably maintains viral replication. Recently, cervical epithelial cell populations that stably maintain HPV-16 replicons at a copy number of approximately 1,000 per cell were derived from an HPV-16-infected patient (W12 cell clone 20863 [W12-E cells]). We used neutral/neutral and neutral/alkaline two-dimensional gel electrophoretic techniques to characterize HPV-16 DNA replication in these cells. When W12-E cells were maintained in an undifferentiated state mimicking the nonproductive stage of the life cycle, HPV-16 DNA was found to replicate primarily by theta structures in a bidirectional manner. The initiation site of HPV-16 DNA replication was mapped to approximately nucleotide 100, and the termination site was mapped to between nucleotides 3398 and 5990. To study the productive stage of HPV-16 DNA replication, W12-E cells were grown under culture conditions that promote differentiation of epithelial cell types. Under these conditions, where virus-like particles were detected, the mode of viral DNA replication changed from theta structure to what is apparently a rolling circle mode. Additionally, CIN 612-9E cells, which were derived from an HPV-31-infected patient and harbor HPV-31 extrachromosomally, exhibited the same switch in the mode of DNA replication upon induction of differentiation. These data argue that a fundamental switch in the mechanism of viral DNA replication occurs during the life cycle of the papillomavirus.

Cell Differentiation↗
PubMed1988

DNA fingerprinting in domestic animals using four different minisatellite probes.

Four probes known to allow DNA fingerprinting in the human (M13, Jeffreys' core sequence, the human alpha globin hypervariable region [HVR], and a mouse probe related to the Drosophila Per gene) were checked for their ability to reveal "genetic bar codes" in cattle, horses, pigs, dogs, chickens, and a European cyprinid fish, the barbel (Barbus barbus L.). Individual-specific patterns were obtained in cattle using M13, Jeffreys' core sequence, and the alpha globin HVR, in horses, dogs, and pigs using M13, Jeffreys' core sequence, and the Per probe, and in chicken and fish using the four different probes. Although we observed a considerable heterogeneity in the extent of interindividual variation, depending on the particular probe-species combination, the fingerprints are polymorphic enough to be used efficiently in animal identification, paternity testing, and as a source of genetic markers for linkage analysis. These markers should substantially accelerate the mapping of genes affecting economically important traits.

Animals↗
PubMed2002

Identification of A-minor tertiary interactions within a bacterial group I intron active site by 3-deazaadenosine interference mapping.

The A-minor motifs appear to be the most ubiquitous helix packing elements within RNA tertiary structures. These motifs have been identified throughout the ribosome and almost every other tertiary-folded RNA for which structural information is available. These motifs utilize the packing of the donor adenosine's N1, N3, and/or 2'-OH against the 2'-OHs and minor groove edge of the acceptor base pair. The ability to identify biochemically which adenosines form A-minor motifs and which base pairs they contact is an important experimental objective. Toward this goal, we report the synthesis and transcriptional incorporation of 5'-O-(1-thio)-3-deazaadenosine triphosphate and its use in Nucleotide Analogue Interference Mapping (NAIM) and Nucleotide Analogue Interference Suppression (NAIS). This analogue makes it possible for the first time to explore the functional importance of the N3 imino group of adenosine in RNA polymers. Interference analysis of the group I self-splicing introns from Tetrahymena and Azoarcus indicates that A-minor motifs are integral to the helix packing interactions that define the 5'-splice site of the intron. Specifically, Azoarcus A58 in the J4/5 region contacts the G.U wobble pair at the cleavage site in the P1 helix, and Azoarcus A167 in the J8/7 region contacts the C13-G37 base pair in the P2 helix. Both of these structural features are conserved between the eukaryotic and bacterial introns. These results suggest that nucleotide analogue interference patterns can identify and distinguish A-minor interactions in RNA tertiary structure, particularly the most prevalent type I and type II varieties. Furthermore, clustering of 3-deazaadenosine interferences is suggestive of A patches, in which a series of consecutive A-minor motifs mediate helix packing. Biochemical identification of these interactions may provide valuable constraints for RNA structure prediction.

Adenosine Triphosphate↗
PubMed1992

Six strains of human immunodeficiency virus type 1 isolated in Japan and their molecular phylogeny.

Five strains of human immunodeficiency virus type 1 (HIV-1) were isolated from five Japanese hemophilia patients. Two isolates, HIV-1[GUN-1] and HIV-1[GUN-2], were from brother patients with hemophilia B and the other three isolates, HIV-1[GUN-3], HIV-1[GUN-4], and HIV-1[GUN-5], were from hemophilia A patients. Another HIV-1 strain, HIV-1[GUN-6], was isolated from a Canadian male homosexual with AIDS. The restriction endonuclease cleavage maps of the proviral genomes of these six HIV-1 strains revealed that they were apparently different from each other. The phylogenetic trees constructed using restriction maps and nucleotide sequences were quite similar, indicating that phylogenetic analyses of Japanese HIV-1 isolates can be done using restriction maps of the proviruses. Phylogenetic analyses showed that they were more closely related to HIV-1s which had been reported to be isolated from homosexual patients in the United States than those isolated from African patients. In particular, GUN-1 and GUN-2 isolates were on the branch of a San Francisco isolate, ARV2, while GUN-5 and GUN-6 isolates were on the branch of HTLV-IIIB-related isolates.

AIDS-Related Complex↗
PubMed2002

The era of genomics: impact on sepsis clinical trial design.

OBJECTIVE: This article aims to address the predictable impact of genetics on the design of clinical trials in the field of critical care medicine, with emphasis on the pathophysiology of sepsis and its treatment. DATA SOURCES: Published articles reporting studies on sepsis and septic shock or assessing the influence of genetics and pharmacogenomics in the treatment of critical illnesses. DATA ANALYSIS: Because most common diseases including sepsis have been shown to be influenced by inherited differences in our genes, completion of the Human Genome Project and the concomitant publication of the human single nucleotide polymorphism map both contribute to change our approach to medicine. Advances in genotyping techniques and bioinformatics enabling detection of single nucleotide polymorphisms have caused an explosion in pharmacogenomics-the research dealing with the interactions of an individual's genotype and the outcome of a drug therapy. Pharmacogenomics will undoubtedly be used to improve future health care and clinical research in different ways. Whereas treatment allocation has been based mainly on phenotype, genetic characterization will help researchers to identify suitable subjects for clinical trials, to facilitate interpretation of the results of clinical trials, and to identify novel targets for future drugs or new markets for current products. As interindividual variability in drug response is a substantial clinical problem, the second major objective of pharmacogenomic research is to decrease adverse responses to therapy through determination of adequate therapeutic targets and genetic polymorphisms that alter drug specificity and toxicity. Ultimately, genetic information will be used to select the most effective therapeutic agent and the optimal dosage to elicit the expected drug response for a given individual. Implementation of genetic criteria for stratification of patient populations and individual assessment of treatment risks and benefits emerges as a major challenge to the pharmaceutical industry. CONCLUSIONS: In the future, technologies such as gene chip array will enhance genetic medicine and provide novel insights into a patient's susceptibility to disease, enabling a better assessment of prognostic risk factors, quicker diagnosis, and accurate prediction of individual responsiveness to drugs. The predictable consequences of such an approach on the prevention and treatment of diseases could revolutionize medicine.

Animals↗
PubMed1993

DNA sequence and transcriptional analyses of the region of the equine herpesvirus type 1 Kentucky A strain genome encoding glycoprotein C.

DNA sequence and transcriptional analyses were performed on the region of the equine herpesvirus type 1 (EHV-1) genome (KyA strain) (map units 0.129 to 0.152) encoding open reading frames (ORFs) 15 and 16. ORF16 encodes a homolog of glycoprotein C of HSV-1 (herpes simplex virus type 1), while ORF15 corresponds in position to HSV-1 UL45 but exhibits no significant homology at the amino acid level. Sequence analyses revealed that the EHV-1 gC ORF of 468 amino acids and ORF15 of 227 amino acids mapped at nucleotides (nt) 716 to 2119 and 2397 to 3077, respectively (relative to the 5' end of the BamHI recognition site at map unit 0.152). ORF15 exhibited significant homology (69% identity) at the amino acid level to the EHV-4 gene located 3' of the EHV-4 gC homolog. Sequence analyses identified potential CAAT and TATA boxes for the EHV-1 gC ORF and a TATA box for ORF15. While no consensus polyadenylation signal was detected between ORFs 15 and 16, two polyadenylation signals were detected 3' of ORF15. Northern blot and S1 nuclease analyses were used to map and characterize the gC and ORF15 mRNAs, and metabolic inhibitors were used to identify the kinetic class of these two genes. The results revealed that gC is a gamma-1 gene which encodes a 2.8-kb mRNA, while ORF15 is a gamma-2 gene encoding a 0.9-kb mRNA which is 3' coterminal with the gC transcript. The gC and ORF15 mRNAs were shown by S1 nuclease analyses to initiate approximately 34 and 26 nucleotides downstream of their respective TATA boxes and to have a common termination site 18 to 20 nucleotides downstream of a consensus polyadenylation signal. Comparative sequence analysis revealed that the KyA strain gC protein differs in only three amino acid residues from the gC protein of the EHV-1 Ab4 and T431 strains, and one of the three amino acid differences occurred within a segment of six contiguous amino acids showing high degree of hydrophilicity in the gC molecule. Further comparative sequence analysis revealed that KyA strain genome has a major deletion in the region of ORF17 which lies 5' of gC in the Ab4 strain. This finding that 1038 base pairs (bp) of the 1203-bp ORF17 is deleted indicates that ORF17 is nonessential for EHV-1 replication in cell culture. To examine the regulation of the EHV-1 gC gene, transient transfection assays using CAT (chloramphenicol acetyltransferase) reporter gene constructs of gC were performed.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2001

Single nucleotide polymorphism markers for genetic mapping in Drosophila melanogaster.

For nearly a century, genetic analysis in Drosophila melanogaster has been a powerful tool for analyzing gene function, yet Drosophila lacks the molecular genetic mapping tools that recently have revolutionized human, mouse, and plant genetics. Here, we describe the systematic characterization of a dense set of molecular markers in Drosophila by using a sequence tagged site-based physical map of the genome. We identify 474 biallelic markers in standard laboratory strains of Drosophila that span the genome. Most of these markers are single nucleotide polymorphisms and sequences for these variants are provided in an accessible format. The average density of the new markers is one per 225 kb on the autosomes and one per megabase on the X chromosome. We include in this survey a set of P-element strains that provide additional use for high-resolution mapping. We show one application of the new markers in a simple set of crosses to map a mutation in the hedgehog gene to an interval of <1 Mb. This new map resource significantly increases the efficiency and resolution of recombination mapping and will be of immediate value to the Drosophila research community.

Animals↗
PubMed1988

Individual specific DNA fingerprints from a hypervariable region probe: alpha-globin 3'HVR.

A probe detecting a hypervariable region (HVR) 3' to the alpha globin locus on chromosome 16 has been used to produce DNA fingerprints. Segregation analysis has revealed multiple, randomly dispersed DNA fragments inherited in a Mendelian fashion with minimal allelism and linkage. The fingerprints are highly polymorphic (probability of chance association between random individuals much less than 10(-14]. The probe is, therefore, a powerful discriminating tool: it is envisaged that this probe will have forensic applications, including paternity cases, and will be informative in linkage analysis.

Alleles↗
PubMed1990

Characterization of the 5' and 3' ends of viral messenger RNAs isolated from BHK21 cells infected with Germiston virus (Bunyavirus).

The 3' ends of the S and M messenger RNAs isolated from BHK21 cells infected with Germiston virus were analyzed by mapping with RNase T2 or nuclease S1. The transcription termination signal was found to be located approximately 115 and 80 nucleotides upstream from the 3' end of the S and M genomic RNA templates, respectively. Both mRNAs were found to possess several adenosine residues at their 3' ends, but were not polyadenylated. They have acquired at their 5' end a heterologous 12- to 18-nucleotide-long sequence, which is not coded for by the virus. Sequencing of the 5' terminal region from single molecules cloned into pBR327 revealed that these primers are rich in C and G residues and possess a U or a C adjacent to the viral sequence.

Animals↗
PubMed1990

Chromosomal footprinting of transcriptionally active and inactive oocyte-type 5S RNA genes of Xenopus laevis.

The chromatin structure of the Xenopus oocyte-specific 5S rRNA genes was examined at high resolution in immature oocyte and somatic cell chromosomes by DNase I footprinting. On oocyte chromatin, where the genes are active, the cleavage preferences over the entire gene region showed a periodic pattern of sensitivity and were dramatically different from the patterns obtained with deproteinized DNA or somatic cell chromatin. Further, the normal binding site for TFIIIA over the internal promoter region was preferentially sensitive to cleavage, indicating that TFIIIA was not bound in the manner predicted by in vitro experiments. In somatic cell chromatin, the oocyte-type 5S genes displayed a cleavage pattern largely similar to deproteinized DNA suggesting the absence of positioned nucleosomes on these inactive genes, although the presence of uncharacterized repressor complexes could not be ruled out. These data are discussed in terms of potential forms of the chromatin structure and alternative mechanisms of oocyte-type gene activation.

Animals↗
PubMed1980

Transcripts of the adeno-associated virus genome: mapping of the major RNAs.

The four major adeno-associated virus type 2 (AAV2)-specific RNAs were mapped on the linear viral genome by a variety of biochemical techniques, including S1 nuclease and exonuclease VII mapping, RNA gel-transfer hybridization, and analysis of reverse transcriptase extension products. All the major AAV2 RNAs were derived from the minus DNA strand and had 3' termini at position 96. The nucleus-specific 4.3- and 3.6-kilobase (kb) RNAs had 5' termini at positions 6 and 19, respectively. The 5' terminus of the 2.6-kb RNA mapped to position 38.5. The predominant 2.3-kb AAV2 mRNA was spliced and contained a short leader sequence (approximately 50 nucleotides) which mapped to position 38.5, coincident with the 5' terminus of the 2.6-kb RNA. The 5' end of the body of the 2.3-kb RNA mapped to position 46.5. These results are discussed in terms of the involvement of single versus multiple promoters (for transcription) and RNA splicing mechanisms in the generation of the AAV2 RNAs.

Chromosome Mapping↗
PubMed1990

Characterization of factors that direct transcription of rat ribosomal DNA.

The protein components that direct and activate accurate transcription by rat RNA polymerase I were studied in extracts of Novikoff hepatoma ascites cells. A minimum of at least two components, besides RNA polymerase I, that are necessary for efficient utilization of templates were identified. The first factor, rat SL-1, is required for species-specific recognition of the rat RNA polymerase I promoter and may be sufficient to direct transcription by pure RNA polymerase I. Rat SL-1 directed the transcription of templates deleted to -31, the 5' boundary of the core promoter element (+1 being the transcription initiation site). The second factor, rUBF, increased the efficiency of template utilization. Transcription of deletion mutants indicated that the 5' boundary of the domain required for rUBF lay between -137 and -127. Experiments using block substitution mutants confirmed and extended these observations. Transcription experiments using those mutants demonstrated that two regions within the upstream promoter element were required for optimal levels of transcription in vitro. The first region was centered on nucleotides -129 and -124. The 5' boundary of the second domain mapped to between nucleotides -106 and -101. DNase footprint experiments using highly purified rUBF indicated that rUBF bound between -130 and -50. However, mutation of nucleotides -129 and -124 did not affect the rUBF footprint. These results indicate that basal levels of transcription by RNA polymerase I may require only SL-1 and the core promoter element. However, higher transcription levels are mediated by additional interactions of rUBF, and possibly SL-1, bound to distal promoter elements.

Animals↗