Characterization of a pro-alpha 2(I) collagen gene mutation by nuclease S1 mapping.
Explore the source record for details and available documents.
Amino Acid Sequence↗
SEARCH · Search PubMed
Results for “Nucleotide Mapping”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Transposon Tn5 mutagenesis to map genes.
Explore the source record for details and available documents.
Cloning, Molecular↗
Messenger RNAs from the transforming region of bovine papilloma virus type I.
Messenger RNAs present in C127 mouse cells transformed by bovine papilloma virus type 1 (BPV-1) were studied by the S1 nuclease protection technique, Northern blotting, and electron microscopic heteroduplex analysis. The results revealed at least five classes of spliced mRNAs which we designate types 1 to 5. They had a common poly(A) addition site located at co-ordinate 53 and all mRNAs, except the type 3 mRNAs, contained an exon located between co-ordinates 41 and 53. In the type 1 mRNAs this exon was connected to a very short leader sequence located around co-ordinate 31. The type 2 mRNAs contained 220 to 400-nucleotide long leaders which were located approximately 1.5 X 10(3) base-pairs further upstream. Two different subclasses of type 2 molecules (2A and 2B) were identified and these had slightly different leaders. The type 4 mRNAs contained a bipartite leader, whereas the type 5 mRNAs carried an approximately 900-nucleotide long leader. The type 3 mRNAs consisted of a main exon located between co-ordinates 32 and 53, linked to the same leader as is present in the type 2A mRNAs. A cap site which presumably is utilized by the type 2A, type 3, type 4 and type 5 mRNAs was mapped at nucleotide 89 in the BPV-1 sequence. A putative cap site for the type 1 mRNAs was mapped at co-ordinate 31.
Animals↗
Hydroxyl radical "footprinting" of RNA: application to pre-mRNA splicing complexes.
We present an adaptation of the hydroxyl radical DNA "footprinting" technique that permits high-resolution mapping of protected regions of RNA. Hydroxyl radical cleaves RNA independently of base sequence and secondary structure of the RNAs examined and allows resolution of protected regions at the single nucleotide level. By using this technique, we show that several regions of the 3' splice site of mRNA precursors are protected during the formation of splicing-specific ribonucleoprotein complexes in an in vitro splicing system. These regions include the 3' intron/exon junction and a portion of the adjacent exon, the polypyrimidine tract, and the site of branch formation. These protections appear to be due to splicing specific complexes since their formation is sensitive to point mutations at crucial residues and requires ATP and incubation. The formation of these protected regions is independent of the presence of a 5' splice site.
Animals↗
Physical and functional map of an amikacin-resistance plasmid isolated from a multiresistant strain of Serratia marcescens.
pTK159, a multiresistance 40-kilobase (kb) plasmid, was isolated from a clinical strain of Serratia marcescens. pTK159 was nonconjugative and carried determinants for resistance to amikacin, streptomycin/spectinomycin, sulfamethoxazole and ampicillin. A physical and functional map of pTK159 was constructed. By cloning various fragments of pTK159 in pACYC184 or pBR322, genes for resistance to amikacin, streptomycin/spectinomycin, and sulfamethoxazole were found to be located on a 2.0-kb BamHI-HindIII fragment, a 1.4-kb HindIII fragment and a 2.1-kb HindIII fragment, respectively. The map of pTK159 was compared with published maps of amikacin-resistance determinants and transposons.
Amikacin↗
Two divergently transcribed genes, soxR and soxS, control a superoxide response regulon of Escherichia coli.
soxR governs a superoxide response regulon that contains the genes for endonuclease IV, Mn2(+)-superoxide dismutase, and glucose 6-phosphate dehydrogenase. The soxR gene encodes a 17-kDa protein; some mutations of this gene cause constitutive overexpression of the regulon. Induction by paraquat (methyl viologen) requires both soxR and a new gene, soxS. soxS is adjacent to soxR, it encodes a 13-kDa protein, and it is required for paraquat resistance. These functions were revealed by studies in which the sequence of the 1.1-kb soxR-soxS region was determined, the 5' ends of the mRNAs were mapped, and complementation tests were performed with soxRS plasmids containing deletions of known sequence. The two genes are divergently transcribed, and the transcripts overlap. The soxS promoter is within the 85-nucleotide intergenic region, whereas the soxR promoter is within soxS. soxS mRNA increases after induction. Both protein products have possible DNA-binding (helix-turn-helix) domains. SoxR contains four cysteines (CX2CXCX5C) that might be part of a sensor region. SoxS shows 17 to 31% homology to the C-terminal portions of members of the AraC family of positive regulators.
Amino Acid Sequence↗
Structural analysis of a hepatitis B virus genome integrated into chromosome 17p of a human hepatocellular carcinoma.
Hepatitis B virus (HBV) is clearly a factor in the development of hepatocellular carcinoma, but its mechanism of action remains obscure. One possibility is that the HBV integration event alters the expression of a nearby growth-regulatory cellular gene. A 9-kilobase (kb) DNA fragment containing an HBV insert plus flanking cellular sequences was cloned from a hepatoma specimen from Shanghai, People's Republic of China. Restriction mapping of the insert revealed a large inverted repeat structure consisting of both viral sequences (encompassing all of the core and pre-S regions and portions of the X and S genes) and at least 3 kb of unique cellular sequences. The virus-cell junction mapped 11 nucleotides from the DR1 region, in a position within the HBV X gene and included in the cohesive overlap region. A probe generated from 1.0 kb of the flanking cellular DNA mapped the viral insert to chromosome 17 in the region designated 17p11.2-17p12, which is near the human proto-oncogene p53. Sequence data from a portion of the flanking cellular DNA revealed a stretch of approximately 70 base pairs that showed highly significant homology with a conserved region of a number of functional mammalian DNAs, including the human autonomously replicating sequence 1 (ARS1).
Base Sequence↗
Mapping of RNA using S1 nuclease and synthetic oligonucleotides.
Explore the source record for details and available documents.
Base Sequence↗
Cloning, structural analysis, and chromosomal localization of the human CSRP2 gene encoding the LIM domain protein CRP2.
The CSRP2 gene encoding the LIM domain protein CRP2 was originally identified in quail based on its strong transcriptional suppression in transformed avian fibroblasts. Here we have isolated a human CSRP2 cDNA clone encoding a 193-amino-acid human CRP2 (hCRP2) protein with 96.4% amino acid sequence identity to the avian homolog. The CSRP2 cDNA clone was used to isolate CSRP2-related clones from gamma EMBL3 and P1 libraries of human genomic DNA. The complete organization of the CSRP2 gene was determined by nucleic acid hybridization, transcriptional mapping, and nucleotide sequence analysis. The gene spans a total of approximately 22 kb and contains six exons. The coding region is confined to exons 2-6 and predicts a hCRP2 protein identical in its amino acid sequence to the protein deduced from the CSRP2 cDNA clone. By fluorescence in situ hybridization using both lambda EMBL3 and P1 library clones as hybridization probes and a new method for computerized signal localization, CSRP2 was mapped to chromosome subband 12q21.1, a region frequently affected by deletion or breakage events in various tumor types. The library screens also led to the isolation of a CSRP2-related pseudogene, CSRP2P, which carried several extensive deletions and nucleotide substitutions but no intervening sequences in comparison to the CSRP2 cDNA sequence. By physical linkage and fluorescence in situ hybridization, CSRP2P was mapped to chromosome subband 3q21.1.
Adaptor Proteins, Signal Transducing↗
Molecular biology and its application in paediatric endocrinology.
Recent advances in molecular biology and genetics have paved the way to a greater understanding of molecular mechanisms of human disease and, in particular, endocrine disorders. For example, new information concerning the structure and function of different receptors and intracellular signalling has allowed precise definition of the molecular defects involved in various disorders such as McCune-Albright syndrome, growth hormone insensitivity syndromes. Although these conditions might be rare, their susceptibility to analysis was due largely to availability and progress of the molecular biological methods. The aim of this review article is to introduce the language of molecular biology by means of a selected group of endocrine disorders.
Base Sequence↗
Molecular analysis of the hemagglutinin genes of Australian H7N7 influenza viruses: role of passerine birds in maintenance or transmission?
In 1985 a fowl plague-like disease occurred in chickens in Lockwood, Victoria, Australia and caused high mortality. An H7N7 influenza virus was isolated from the chickens (A/Chicken/Victoria/1/85); additionally, an antigenically similar virus was isolated from starlings (A/Starling/Victoria/5156/85) and serological evidence of H7N7 virus infection was found in sparrows. Antigenic analysis with monoclonal antibodies to H7, oligonucleotide mapping of total vRNA, and sequence analysis of the HA genes established that the chicken and starling influenza viruses were closely related and probably came from the same source. There was high nucleotide sequence homology (95.3%) between the HA genes of A/Chick/Vic/85 and a fowl plague-like virus isolated from chickens in Victoria 9 years earlier [A/Fowl/Vic/76 (H7N7)]. The sequence homologies indicated that the A/Chick/Vic/85 and A/Fowl/Vic/76 were derived from a common recent ancestor, while another recent H7N7 virus, Seal/Mass/1/80 originated from a different evolutionary lineage. Experimental infection of chickens and starlings with A/Chick/Vic/1/85 (H7N7) was associated with high mortality (100%), transmission to contact birds of the same species, and virus in all organs. In sparrows one-third of the birds died after infection and virus was isolated from most organs; transmission to contact sparrows did not occur. In contrast, the H7N7 virus replicated in ducks and spread to contact ducks but caused no mortality. These studies establish that the host species plays a role in determining the virulence of avian influenza viruses, and provide the first evidence for transmission of virulent influenza viruses between domestic poultry and passerine birds. They support the hypothesis that potentially virulent H7N7 influenza viruses could be maintained in ducks where they cause no apparent disease and may sometimes spread to other wild birds and domestic poultry.
Amino Acid Sequence↗
A study of the evolution of coxsackievirus A24 variant in Ghana by viral RNA fingerprinting analysis.
An epidemic of acute haemorrhagic conjunctivitis (AHC) caused by coxsackievirus A24 variant (CA24v) was reported in Accra, Ghana in May 1987. We studied 7 of the viral strains collected from May to November, 1987, by RNA genome fingerprinting. Pairwise comparisons of the oligonucleotide maps showed that genetic similarity among them ranged from between 60.0 to 84.7%. Using base sequence variations deduced from genetic similarity among the isolates, isolation time of the strains and the rate of nucleotide substitution (estimated in a previous paper, Miyamura et al., 1990), we calculated divergence times and constructed a phylogenetic tree. This tree indicated that all the 7 strains had diverged from each other from 11 to 26 months before the AHC epidemic in Accra. CA24v may have been introduced into the country or the neighbouring area, at least, more than two years earlier, i.e. in the early half of 1985.
Adult↗
The use of T1 oligonucleotide mapping to determine that the emergence of Canadian 1973-like and 1974-like H3N2 strains in 1984 was the result of laboratory contamination.
Influenza undergoes extensive antigenic variation in nature. These new antigenic variants invariably supplant the preceding antigenic type that then disappears. In apparent contrast to this, two H3N2 strains that were forwarded to the Laboratory Centre for Disease Control, Ottawa, in 1984 from a Canadian public health laboratory for reference analysis were shown to be most closely related to 1973 and 1974 strains. Laboratory contamination on isolation by the regional public health laboratory was the most likely explanation for the occurrence of these strains, since one of the isolates (RV/76/84) was identical by T1 mapping to an A/Eng-like isolate being grown in the laboratory of isolation. The two isolates, RV/74/84 and RV/76/84, were shown to be distinct from each other and from prototype H3N2 strains by RNase T1 oligonucleotide mapping, SDS-PAGE, peptide mapping of hemagglutinin, and hemagglutination inhibition assay. RV/74 and RV/76 appeared to have been genetically stable for the 10 to 11 years preceding 1984; this is consistent with laboratory frozen storage for this period of time. This paper demonstrates the utility of RNase T1 mapping for the characterization of novel or spurious influenza isolates.
Humans↗
Implications of a functional large ribosomal RNA with only three modified nucleotides.
The sequence and structure of the peptidyl transferase region of large subunit ribosomal RNA is highly conserved and specific modified nucleotides could be important structural or functional elements in the catalytic center responsible for peptide bond formation. In fact, it has not been possible to reconstitute active E coli 50S subunits from in vitro transcripts of 23S rRNA and total 50S proteins. It is significant therefore, that the PET56 gene of yeast encodes an essential ribose methyltransferase that specifically modifies a universally conserved nucleotide, G2270, in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S). Since the loss of this modification in yeast mitochondrial 21S rRNA severely affects the assembly of 54S subunits, it is likely that the analogous 2'-O-methylguanosine at position 2251 (Gm2251) in E coli 23S rRNA is also required for the assembly of 50S subunits. Gm could be a critical structural determinant for the correct folding of the rRNA, the binding of one or more ribosomal proteins, or the interaction of the rRNA with tRNA. Previous work has shown that the mitochondrial large rRNAs are minimally modified relative to the E coli and eukaryotic cytoplasmic rRNAs. By direct chemical analysis using combined high performance liquid chromatography-mass spectrometry, the modification status of the yeast mitochondrial rRNAs was reexamined, revealing the presence of Gm, Um and pseudouridine (psi) in 21S rRNA. The Um was mapped to nucleotide 2791, which corresponds to the ribose methylated and universally conserved U2552 in E coli 23S rRNA, and the psi has been recently mapped to position 2819, which corresponds to psi 2580 in E coli 23S rRNA. The retention of Um and psi nucleotides in the peptidyl transferase center of the otherwise minimally modified mitochondrial rRNAs suggests that these modifications, like Gm2270, might be essential for ribosome assembly or function or both.
Base Sequence↗
A new approach to mapping co-localization of multiple glycosyl transferases in functional Golgi preparations.
We have developed a new method to co-localize multiple glycosyl transferases in different Golgi compartments. The approach relies on the proven ability of intact, sealed rat liver Golgi preparations to concentrate exogenous labeled sugar nucleotides into the lumen where they glycosylate either endogenous or artificial acceptors. The premise is that if two glycosyl transferases are co-localized within the same compartment, they will compete for the limited amount of transported donor. If the donor is consumed in glycosylating a permeable artificial glycoside within a Golgi compartment, it will be unavailable to glycosylate endogenous products within that same compartment. The greater the degree of transferase co-localization, the greater the potential decrease in glycosylation of endogenous acceptors. We provide an example consistent with these predictions. Adding 1 microM UDP[3H]Gal to Golgi preparations followed by a chase with a cocktail of unlabeled sugar nucleotides labels mostly endogenous N-linked oligosaccharides containing both beta 1,3- and beta 1,4[3H]Gal residues with and without sialic acid. Addition of increasing amounts of 4-methylumbelliferyl-beta-xyloside (Xyl beta MU) produces [3H]Gal1 beta, 4Xyl beta MU and leads to a reciprocal decrease in labeling of a restricted set of the endogenous acceptors. This decrease is preferential for [3H]Gal beta 1-->3GlcNAc beta 1-->R and, to a lesser extent, [3H]Gal beta 1-->4GlcNAc beta 1-->R structures in neutral and mono-sialylated oligosaccharides; synthesis of these structures in di- and tri-sialylated oligosaccharides was unaffected. These preferential decreases are not seen in detergent permeabilized, sugar nucleotide transport-independent Golgi incubations, and are not due to inhibition by the Gal beta 1,4Xyl beta MU product. These results argue that there is significant overlap in the functional co-localization of sialyl and galactosyltransferases in rat liver Golgi preparations and that GAG chain core specific Galactosyltransferase I is co-localized with subsets of N-glycan Gal beta 1,3 and Gal beta 1,4 transferases. This approach can be used with other glycosides and sugar nucleotides to map and co-localize other glycosyl transferases. The functional compartments defined by this approach may or may not correspond entirely with morphologically defined Golgi domains.
Animals↗
Analysis of African cassava mosaic virus recombinants suggests strand nicking occurs within the conserved nonanucleotide motif during the initiation of rolling circle DNA replication.
Intact clones containing partial repeats of the genomic components of African cassava mosaic (ACMV DNAs A and B) are infectious when mechanically coinoculated onto Nicotiana benthamiana. Monomeric genomic components may be generated either by homologous recombination or, when two copies of the origin of replication (ori) are present, by a modified rolling circle replication mechanism in which nascent single-stranded DNA is resolved by the introduction of nicks at both oris. DNA B partial repeats with duplicated common region sequences containing combinations of wild-type sequences and nonlethal mutations at nucleotides 151 and 155 within the putative stem-loop region have been constructed and introduced into plants in the presence of DNA A. Analysis of progeny indicates that monomers are generated by DNA strand nicking preferentially between nucleotides 151 and 155, suggesting a nonrandom replicative release mechanism involving the ubiquitous TAATATTAC motif (nucleotides 146-154). Viable ACMV DNA A deletion mutants are known to revert to wild-type size during systemic infection by generating tandem repeats. The recombination point in one such revertant has been mapped between nucleotides 152 and 153. Just as ori-nicking enzymes mediate recombinational events during prokaryotic rolling circle DNA replication, the result suggests that a nick has been introduced in the virion-sense strand within the nonanucleotide motif (TAATATT decreases AC) during the initiation of ACMV DNA replication.
Base Sequence↗
Escherichia coli Tus protein acts to arrest the progression of DNA replication forks in vitro.
A polar DNA replication barrier is formed when the DNA-binding protein Tus forms a complex with any of the four 23-base-pair terminator (ter) sites found in the terminus region of the Escherichia coli chromosome. We have used a plasmid DNA replication system reconstituted with purified proteins in vitro to investigate the interaction of the Tus protein with the replication fork. Purified Tus protein alone is necessary and sufficient to arrest DNA replication on ColE1-type plasmid templates containing ter sites. Tus protein-catalyzed termination depends upon the orientation of the ter site in the plasmid DNA. Nucleotide resolution mapping of the terminated nascent DNA shows that leading-strand DNA synthesis arrests at the point of contact with the Tus protein, while the final lagging-strand primer sites are 50-70 nucleotides upstream. In addition, the distribution of leading-strand arrest sites changes when the composition of the proteins on the lagging-strand side of the replication fork is altered.
Bacterial Proteins↗
Human glucokinase gene: isolation, structural characterization, and identification of a microsatellite repeat polymorphism.
The gene encoding human glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), a major component of glucose sensing in pancreatic islet beta-cells, was isolated and characterized. The gene was shown by Southern blotting to exist as a single copy in the genome which mapped to chromosome 7p. It contained 12 exons including two tissue-specific first exons, one active in islet beta-cells (1B), and the other active in liver (1H), and one optional cassette exon which was expressed as a minor form in the liver. Thus the three previously reported isoforms of glucokinase mRNA were the result of tissue-specific activation of separate liver and islet promoters and subsequent alternative splicing events. Eleven exons, including 1H and the optional cassette exon 2A, were scattered over 16 kilobase (kb) in the genome, while exon 1B was separated from the rest by at least 20 kb. Although the islet promoter was found to lack a TATA box, a major transcript from the islet promoter was mapped 486 nucleotides upstream of the translation initiation site. The presence in the islet glucokinase promoter of the potential control element GCCACCAG, a homology of the regulatory element present in both human insulin (GCCACCGG) and rat insulin (GCCATCTG) genes, implied a possible tissue-specific regulatory role of this element. The liver promoter was found to contain a TATA box-like sequence, and transcription was initiated predominantly at 168 nucleotides upstream of the translation initiation site of the major isoform. A new highly polymorphic microsatellite, composed of a compound imperfect dinucleotide repeat [GT]15[GA]8CA[GA]7CA[GA]3AA[GA]2, was mapped 6 kb upstream of islet exon 1. A polymerase chain reaction-based assay was developed, and seven different sized alleles were identified in American Blacks. The sequence information reported here, along with the new polymorphic marker, will make it possible to clarify the molecular basis of potential glucokinase defects in noninsulin-dependent diabetes mellitus patients and may further elucidate the nature of genetic susceptibility to the development of this common metabolic disease.
Amino Acid Sequence↗