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PubMed1990

Rearrangement of VHa1-encoding Ig gene segment to the a2 chromosome in an a1/a2 heterozygous rabbit. Evidence for trans recombination.

VDJ genes were cloned from leukemic B cells of an a1/a2 heterozygous Emu-cmyc transgenic rabbit. Restriction mapping and nucleotide sequence analysis indicated that one clone, 5C3, had a VHa1-encoding gene segment functionally rearranged to a JH gene segment from the a2 chromosome. This VDJ gene may be the result of a trans recombination between a VH gene on the a1 chromosome and a JH gene segment on the a2 chromosome or, it may be the result of a cis recombination if the a2 chromosome contains VHa1-encoding gene segments.

Amino Acid Sequence↗

PubMed1990

Physical mapping of two temperature-sensitive adenovirus mutants affected in the DNA polymerase and DNA binding protein.

We have determined the exact nature of two thermosensitive (ts) adenovirus mutants, H5ts19 and H5ts149, which map to different genes in the E2 transcription unit. The H5ts19 mutation appears to stem from a single base-pair change of A-T to G-C at position 1840 (numbering as in ref. 1), corresponding to codon 154 of the gene coding for DBP. This results in a glutamine-to-arginine change in the amino-terminal domain of the protein. H5ts19 is defective in a late stage of infection, during virus assembly. This phenotype strongly differs from that described for the limited number of known DBP mutants, indicating that DBP is not only functional during DNA replication, but also plays a role in the late phase of the infection cycle. The defect of the (N group) mutant H5ts149 affects the initiation of viral DNA replication. Marker rescue experiments followed by nucleotide sequence analysis of H5ts149 DNA revealed a single point mutation in the gene coding for the Ad pol. A transition of C-G to A-T at position 7563 (numbering as in ref. 2) changes amino acid residue 411 of Ad pol, a leucine residue, to phenylalanine. This mutation is located in a region conserved among various DNA polymerases, which suggests an important role of this domain in DNA replication.

Adenoviridae↗

PubMed1988

Regulation of proline utilization in Salmonella typhimurium: molecular characterization of the put operon, and DNA sequence of the put control region.

The two genes required for proline utilization (put) in Salmonella typhimurium form a divergent operon. Extensive genetic evidence suggests that transcription of the put operon is autoregulated by the putA gene product, a membrane-associated dehydrogenase. In order to understand the mechanism of regulation, we characterized plasmid clones of the put operon. A 7.5 kb clone contains both of the put structural genes and regulatory sites. This clone only expressed two unique proteins corresponding to the putA and putP gene products. By comparing the physical and genetic maps of the put operon, the position of the put regulatory region was defined and the DNA sequence of this region was determined. Analysis of the DNA sequence indicated several potential regulatory sites for the put genes. Based on genetic and physical mapping studies, the most likely regulatory sites are two convergent promoters approximately 30 bp apart. A 27 bp palindrome located between the two promoters may be the operator for autoregulation by the PutA protein. The putA translational start site is 40 bp downstream of its putative mRNA start site. The putP promoter and its translational start site are separated by a 400 bp untranslated region.

Base Sequence↗

PubMed1988

Complementation of a truncated membrane-bound Enzyme IINag from Klebsiella pneumoniae with a soluble Enzyme III in Escherichia coli K12.

Cloning and analysis of the gene nagE encoding Enzyme IINag (EIINag) from Klebsiella pneumoniae revealed strong similarities with the corresponding gene from Escherichia coli K12. Truncated EIINag proteins were generated by inserting a series of Tn1725 transposons into the structural gene; the positions of the insertions were mapped by restriction enzyme analysis, and the activity of the polypeptides determined by in vitro and in vivo tests. Insertions in the region encoding the amino-terminal half of the protein invariably abolished transport and phosphorylation activity, while truncated proteins lacking a C-terminal domain homologous to the soluble Enzyme III (crr gene) could be complemented by this molecule to nearly wild-type activity.

Cell Membrane↗

PubMed1987

Physical comparison of parathion hydrolase plasmids from Pseudomonas diminuta and Flavobacterium sp.

Restriction maps of two plasmids encoding parathion hydrolase have been determined. pPDL2 is a 39-kb plasmid harbored by Flavobacterium sp. (ATCC 27551), while pCMS1 is a 70-kb plasmid found in Pseudomonas diminuta (strain MG). Both plasmids previously have been shown to share homologous parathion hydrolase genes (termed opd for organophosphate degradation) as judged by DNA-DNA hybridization and restriction mapping. In the present study, we conducted DNA hybridization experiments using each of nine PstI restriction fragments from pCMS1 as probes against Flavobacterium plasmid DNA. The opd genes of both plasmids are located within a highly conserved region of approximately 5.1 kb. This region of homology extends approximately 2.6 kb upstream and 1.7 kb downstream from the opd genes. No homology between the two plasmids is evident outside of this region.

Aryldialkylphosphatase↗

PubMed1989

Rapid chemical mapping of dengue virus variability using RNA isolated directly from cells.

Osmium tetroxide and hydroxylamine (in combination with piperidine) have previously been shown to cleave mismatched T and C bases, respectively, in DNA.DNA heteroduplexes. In this work we report that mismatched T and C bases were similarly cleaved in DNA.RNA heteroduplexes of nucleic acids derived from different strains of dengue virus type 2. Further, some matched T or C bases one or two bases from mismatches were also chemically reactive and thus cleavable as detected by minor bands. Cleavages both at and near mismatches combined to generate a simply obtained pattern of difference between virus strains that could be used as a fingerprint of a given virus relative to another. The patterns obtained using viral RNA of one strain hybridized with the cDNA of another were similar for RNA prepared from purified virions and for total RNA extracted from infected cells. Use of probes of both senses should detect all differences. Two sequenced (NGC and PUO-218) and one unsequenced (D80-100) strains of virus were compared in these studies. The analyses allowed proof reading of the differences between NGC and PUO-218, ascertained from nucleotide sequencing, and demonstrated that D80-100 is more similar to PUO-218 than to NGC.

Base Composition↗

PubMed1990

Mapping of the prekallikrein-binding site of human H-kininogen by ligand screening of lambda gt11 expression libraries. Mimicking of the predicted binding site by anti-idiotypic antibodies.

High molecular weight (H-)kininogen, a non-enzymatic cofactor of the contact activation system, has on the COOH-terminal part of its light chain a unique binding site which complexes prekallikrein or factor XI with high affinity and specificity. In a conventional protein fragmentation approach, the prekallikrein-binding site was mapped to positions 556-595 of the human H-kininogen sequence (Tait, J. F., and Fujikawa, K. (1986) J. Biol. Chem. 261, 15396-15401). To gain more insight into the minimum structural requirements of the prekallikrein-binding site, we have developed an alternative strategy employing the lambda gt11 expression cloning system. A ligand assay was established which probes for the binding site in H-kininogen or recombinant fusion proteins thereof by complexation with prekallikrein, followed by a specific antibody against prekallikrein and a secondary labeled antibody. A cDNA library constructed in lambda gt11 from random fragments of a cDNA clone encoding the COOH-terminal part of the kininogen light chain was screened by the ligand assay, and 17 positive clones were identified. Analysis of their inserted cDNA sequences revealed a consensus sequence of 119 nucleotides which maps to the extreme 3' end (positions 1759-1877) of the coding part of the prekininogen mRNA. The consensus sequence encodes positions 569-607 of the kininogen light chain and overlaps by 27 residues (positions 569-595) with the binding segment identified previously by the fragment approach. Analysis of successively shortened peptides revealed that the common segment of 27 residues but not truncated versions thereof contains the essential structural elements for prekallikrein binding. This conclusion was corroborated by the finding that anti-idiotypic antibodies toward a monoclonal antibody directed to the binding segment of 27 residues bear internal image(s) of the binding site of H-kininogen. It is pointed out that the methodology described in this study may prove generally useful in the cloning and mapping of high affinity binding sites of proteins.

Amino Acid Sequence↗

PubMed1988

Mapping functional regions of transcription factor TFIIIA.

Functional deletion mutants of the trans-acting factor TFIIIA, truncated at both ends of the molecule, have been expressed by in vitro transcription of a cDNA clone and subsequent cell-free translation of the synthetic mRNAs. A region of TFIIIA 19 amino acids or less, near the carboxyl terminus, is critical for maximal transcription and lies outside the DNA-binding domain. The elongated protein can be aligned over the internal control region (ICR) of the Xenopus 5S RNA gene with its carboxyl terminus oriented toward the 5' end of the gene and its amino terminus oriented toward the 3' end of the gene. The nine "zinc fingers" and the linkers that separate them comprise 80% of the protein mass and correspond to the DNA-binding domain of TFIIIA. The zinc fingers near the amino terminus of the protein contribute more to the overall binding energy of the protein to the ICR than do the zinc fingers near the carboxyl end. The most striking feature of TFIIIA is its modular structure. This is demonstrated by the fact that each zinc finger binds to just one of three short nucleotide sequences within the ICR.

Animals↗

PubMed1986

A 'Southern Cross' method for the analysis of genome organization and the localization of transcription units.

A 'Southern Cross' hybridization method is described which permits the rapid restriction mapping of DNA molecules, up to 40 kb in size, for at least ten enzymes in a single operation. The procedure allows the full set of 32P-end-labelled fragments derived from one restriction enzyme digest to intersect and attempt to hybridize to the gel-separated fragments of as many as ten unlabelled digests immobilized on parallel sheets of filter paper. A two-dimensional array of hybridization spots is revealed on each recipient paper, indicating which radioactive and non-radioactive DNA fragments have sequences in common. A restriction map can then be directly and simply deduced from the matrix of hybridization spots in each cross-blot. The method affords advantages over other procedures for obtaining restriction maps in terms of the time required, the number of restriction enzymes that can be mapped, and the potential for eliminating ambiguity. It is also sufficiently sensitive to detect DNA rearrangements and restriction-site polymorphisms in moderately complex genomes. Furthermore, the procedure is applicable to other aspects of the study of genome organization: for example, the exon and intron areas of a segment of cloned genomic DNA can be identified by cross-hybridizing a set of radioactive restriction fragments from the genomic clone against immobilized RNA from a cell type of interest.

DNA Restriction Enzymes↗

PubMed1987

Mapping and sequencing of a gene from myxoma virus that is related to those encoding epidermal growth factor and transforming growth factor alpha.

Myxoma virus, a Leporipoxvirus and agent of myxomatosis, was shown to possess a gene with the potential to encode an epidermal growth factorlike factor. Its relationship to other members of this family, including the poxvirus growth factors from Shope fibroma virus and vaccinia virus, was analyzed. Alignment of DNA sequences and related open reading frames of myxoma virus and Shope fibroma virus indicated colinearity of genes between these poxviruses.

Amino Acid Sequence↗

PubMed1993

TAP2 gene polymorphism segregates with DR-DQ in DR/DP recombinant siblings.

RING 11, a second transport-associated gene (TAP2), has been recently identified in the DR-DP interval of the human class II region. Two predominant alleles, TAP2A and TAP2B, differing by 17 amino acids at the C-terminus of the ATP-binding domain are present in the Caucasoid population at frequencies of 79% and 21%, respectively. In the rat, polymorphism of the TAP genes were found to influence peptide loading of MHC class I molecules and, in humans, it was speculated that variation in peptide loading of HLA-B27 molecules might be also linked to factors altering antigen presentation presumably encoded in the HLA region. To determine whether TAP2 gene polymorphism may be relevant to peptide loading in humans, we typed 41 HLA-ABC, DR-identical pairs for TAP2A and TAP2B by PCR-SSO hybridization or direct genomic sequencing. In eight cases, GLO-different and, in six cases, DP-different recombinant siblings were included. Allele frequencies for TAP2A and TAP2B were as previously reported (74% and 26%, respectively). In all pairs, TAP2 gene polymorphism segregated with the DR-DQ type, mapping the TAP2 gene telomeric to the recombination hot spot in the DR-DP interval of the human class II region. We conclude that, in HLA-identical siblings, TAP2 gene differences are very unlikely to occur. Thus, in HLA-identical siblings, minor histocompatibility antigenic differences cannot be attributed to variant peptide loading due to TAP2 gene polymorphism.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR.

BACKGROUND: Insertion sequences (IS) are small DNA segments capable of transposing within and between prokaryotic genomes, often causing insertional mutations and chromosomal rearrangements. Although several methods are available for locating ISs in microbial genomes, they are either labor-intensive or inefficient. Here, we use vectorette PCR to identify and map the genomic positions of the eight insertion sequences (IS1, 2, 3, 4, 5, 30, 150, and 186) found in E. coli strain CGSC6300, a close relative of MG1655 whose genome has been sequenced. RESULTS: Genomic DNA from strain CGSC6300 was digested with a four-base cutter Rsa I and the resulting restriction fragments ligated onto vectorette units. Using IS-specific primers directed outward from the extreme ends of each IS and a vectorette primer, flanking DNA fragments were amplified from all but one of the 37 IS elements identified in the genomic sequence of MG1655. Purification and sequencing of the PCR products confirmed that they are IS-associated flanking DNA fragments corresponding to the known IS locations in the MG1655 genome. Seven additional insertions were found in strain CGSC6300 indicating that very closely related isolates of the same laboratory strain (the K12 isolate) may differ in their IS complement. Two other E. coli K12 derivatives, TD2 and TD10, were also analyzed by vectorette PCR. They share 36 of the MG1655 IS sites as well as having 16 and 18 additional insertions, respectively. CONCLUSION: This study shows that vectorette PCR is a swift, efficient, reliable method for typing microbial strains and identifying and mapping IS insertion sites present in microbial genomes. Unlike Southern hybridization and inverse PCR, our approach involves only one genomic digest and one ligation step. Vectorette PCR is then used to simultaneously amplify all IS elements of a given type, making it a rapid and sensitive means to survey IS elements in genomes. The ability to rapidly identify the IS complements of microbial genomes should facilitate subtyping closely related pathogens during disease outbreaks.

Bacterial Typing Techniques↗

PubMed1987

Demonstration of an altered phenylalanyl-tRNA synthetase in an analogue-resistant mutant of Aspergillus nidulans.

We have isolated and characterized a new class of p-fluorophenylalanine (FPA)-resistant mutant in Aspergillus nidulans using a phenA strain as the wild type, by optimizing the conditions of growth. All four spontaneous mutants selected on a medium containing FPA were found to be recessive to their wild-type alleles in heterozygous diploids. Complementation analyses and linkage data showed that they were allelic and mapped at a single locus (fpaU) in the facA-riboD interval on the right arm of linkage group V. Partial purification and characterization of Phe-tRNA synthetase from wild-type and mutant strains revealed that the mutant enzyme had a greatly reduced ability to activate the analogue. It is suggested that mutation in the fpaU gene brings about a structural alteration in Phe-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗

PubMed1990

Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III.

The replication frequency of IncFII plasmids is regulated through the availability of a rate-limiting protein, RepA. The synthesis of this protein is controlled post-transcriptionally by a small antisense RNA, CopA, which binds to the leader region of the RepA mRNA (CopT). In this communication we report studies of the IncFII plasmid R1. We show that the duplex between CopA and CopT is cleaved specifically in vivo. The in vivo cleavage maps to the same position as that resulting from in vitro cleavage of a CopA/CopT duplex by purified RNase III. By introducing plasmids carrying translational repA-lacZ fusions into cells deficient in RNase III we show that the expression of repA is elevated when RNase III activity is severely decreased. Hence, cleavage by RNase III seems to be a key event in the copy number control system of plasmid R1.

Bacterial Proteins↗

PubMed1986

Characterization of three HLA-DR beta genes isolated from an HLA-DR 3/4 insulin-dependent diabetic patient.

Three HLA-DR beta genes were isolated from a Swedish HLA-DR3/4 insulin-dependent diabetes mellitus (IDDM) patient and characterized by restriction endonuclease mapping and nucleotide sequence analysis. Two out of the three DNA sequences differed from those of published DR beta-chain sequences. A DR beta-gene probe prepared from exon 4 and flanking sequences was used in a Southern blot analysis of blood donors' DNA and DNA from HLA-DR3/4 IDDM patients and HLA-DR-matched healthy control subjects. This probe differentiated HLA-DR3/4 IDDM patients and HLA-DR-matched controls in the Scandinavian population but not in the North American Caucasoid population.

Amino Acid Sequence↗

PubMed1990

Localization of DNA protein-binding sites in the proximal and distal promoter regions of the mouse alpha-fetoprotein gene.

DNase I footprinting assays were performed to identify the binding sites for putative trans-acting factors involved in the control of alpha-fetoprotein (AFP) gene expression using mouse AFP promoter fragments (-839 to +56) and nuclear protein extracts from fetal, newborn, and adult livers and from brain and kidney. Our studies have shown that with nuclear protein from adult mouse liver, there are 14 protected regions in the AFP promoter up to -839 base pairs (bp). Region I (-82 to -43) was protected by at least three different factors, one of which is CCAAT-binding/enhancer-binding protein. This region is highly conserved in the mouse, rat, and human AFP genes and has been shown previously to be essential for the regulation of tissue-specific expression in mouse. Differences in DNase I protection with fetal, newborn, and adult nuclear proteins have been observed in the proximal promoter region (up to -202 bp) and in regions further upstream (up to -839 bp). Significant differences among liver, kidney, and brain nuclear protein-binding sites have also been observed. In these studies, we have mapped the fetal and adult nuclear protein-binding sites of the cis-acting DNA sequences of the mouse AFP proximal promoter (up to -200) and have identified specific protein-binding sites in the distal promoter (-200 to -839). We have also identified the sites of the AFP promoter which bind nuclear proteins from highly differentiated tissues in which AFP is not expressed.

Aging↗

PubMed1990

Distinct vascular and intestinal smooth muscle myosin heavy chain mRNAs are encoded by a single-copy gene in the chicken.

We examined whether the gizzard MHC gene is expressed in other smooth muscle tissues and, if so, whether there exist any smooth muscle MHC isoforms at the mRNA level. Northern blot analysis showed that the gizzard MHC gene was also expressed in the aorta and jejunum, but not in the pectoralis muscle or in fibroblasts. This indicates that striated muscle and non-muscle MHC isoforms are encoded in genes distinct from the smooth muscle MHC gene. Further, nuclease S1 mapping showed that the aortic smooth muscle MHC mRNA was distinct from the gizzard mRNA in the 5'-terminal coding region. Both of these mRNA species are expressed in the jejunum. These observations suggest that there exist at least two chicken smooth muscle MHC isoforms, vascular-type and intestinal-type, and that these isoforms are generated from a single-copy gene, probably by an alternative mRNA processing mechanism.

Animals↗

PubMed1987

Sequence and S1 nuclease mapping of the 5' region of the dihydrofolate reductase-thymidylate synthase gene of Leishmania major.

The 5' structure of mRNA transcribed from the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene of the protozoan parasite Leishmania major has been characterized. S1 nuclease mapping identifies a heterogenous 5' structure which is not affected by growth phase or developmental stage. The DNA sequence of the 5' region of the DHFR-TS gene does not reveal homology with other trypanosomatid genes, eukaryotic consensus genetic elements, or the mammalian DHFR promoter element. This latter finding is especially significant as we show that the 5' region of the E. coli DHFR gene exhibits homology to the mammalian DHFR promoter element, despite their greater evolutionary distance.

Animals↗