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At least 937 records · Page 52Linked to original sources
PubMed1988

Instability of recombinant pUB110 plasmids in Bacillus subtilis: plasmid-encoded stability function and effects of DNA inserts.

Two series of pUB110-derived plasmids were constructed to study segregational stability in Bacillus subtilis. pEB plasmids were based on the entire pUB110, whereas pLB plasmids lack the membrane-binding areas BA3 and BA4. Two kinds of stability defects were observed. The first was characterized by a strong size dependency and occurred with different inserts at various positions in pLB and pEB plasmids. Size-dependent reductions in plasmid copy numbers appeared to underly this phenomenon. This may render pUB110 unsuitable for the cloning of inserts larger than about 3 kb, in particular if no selective conditions can be applied. The second defect, observed with pLB plasmids, was caused by the absence of the membrane-binding areas BA3 and BA4. Deletion of BA3 resulted in the accumulation of single-stranded plasmid DNA, suggesting that BA3 contains the initiation signal for complementary strand synthesis. The BA3 region is very rich in hyphenated dyad symmetry which, in single-stranded DNA, could result in several stable alternative secondary structures. It is speculated that the activity of the BA3-associated initiation signal contributes to the segregational stability of pUB110-derived plasmids in B. subtilis. The absence of the BA3 stability function could not account for all stability defects observed. Additional stability functions seemed to be located on the BA4 fragment.

Bacillus subtilis↗
PubMed1989

The complete nucleotide sequence of a small cryptic plasmid from Lactobacillus plantarum.

The complete nucleotide sequence of a cryptic plasmid isolated from a Lactobacillus plantarum strain has been determined. The plasmid, designated pC30i1, has a molecular size of 2140 bp and a GC content of 37%. The sequence contains one major open reading frame (ORF R) of 951 bp, encoding a basic polypeptide of 317 amino acids, and a molecular weight of 36,956. ORF R shows extensive sequence similarity with genes coding for replication-associated proteins in a group of gram-positive plasmids known to replicate via single-stranded intermediates (ssDNA plasmids), and a stretch of 9 amino acids in the translation of ORF R closely matches a conserved region in these proteins, as well as the active site of the phi X174 Rep protein. Sequences similar to the ssDNA plasmid origins of replication are also present in the pC30i1 sequence, strengthening the hypothesis that pC30i1 belongs to the ssDNA plasmid family. The other main feature of the pC30i1 sequence is a noncoding region consisting of 14 direct, imperfect repeats of a 17-bp sequence, which may have an incompatibility function.

Amino Acid Sequence↗
PubMed1992

Purification and cDNA cloning of a novel factor produced by a human T-cell hybridoma: sequence homology with animal lectins.

We have purified a novel immunoregulatory factor (BMPG: bone-marrow proteoglycan) produced by a T-cell hybridoma, with a monoclonal antibody column. Using an oligonucleotide probe corresponding to the partial amino acid sequence of BMPG, we cloned, sequenced, and expressed a cDNA for BMPG. BMPG has 222 amino acid residues with a 16 N-terminal signal sequence, so the mature form has 206 amino acid residues. BMPG was found to have unique characteristics: it has three types of sugar chains and it shows a marked homology with animal lectins including the human asialoglycoprotein receptor, chicken hepatic lectin and the homing receptor of lymphocytes.

Amino Acid Sequence↗
PubMed1992

Sequence and chromosome assignment to 11p13-p12 of human RAG genes.

The recombination-activating genes RAG-1 and RAG-2 are required for V(D)J DNA rearrangements at loci for immunoglobulin and T cell receptor genes. We isolated the human RAG-2 gene and determined its nucleotide sequence. Mapping analysis of RAG-1 and RAG-2 genes on human chromosomes by fluorescence in situ hybridization indicated that the genes are located on chromosome 11p13-p12. RAG-1 and RAG-2 do not seem to be linked to any of the primary immunodeficiencies for which defective genes have already been mapped.

Amino Acid Sequence↗
PubMed1986

Rapid method for the identification and screening of herpesviruses by DNA fingerprinting combined with blot hybridization.

Rapid characterization of herpesvirus isolates exemplified by equine herpesviruses is described. Total DNAs were isolated from virus infected small scale cell cultures. The DNA fragments obtained after restriction enzyme digestion were separated on agarose gels, transferred and immobilized on filter membranes. A radioactively labelled probe derived from the purified DNA of an EHV-1 reference strain was used for hybridization in order to detect the restriction fragments of different EHV-1 field isolates. This method allows the typing of many isolates within a short period of time.

Animals↗
PubMed1989

A simplified procedure for oligonucleotide fingerprint analysis of multiple bluetongue virus genome segments utilizing small gels.

The technique of oligonucleotide fingerprinting is a useful tool for analyzing sequence homology among RNA molecules. A rapid method for the simultaneous production of multiple fingerprints has been developed using a commercially available electrophoresis apparatus. The system makes use of relatively small gels, yielding fingerprints that when compared to conventional systems are of reduced size but of comparable resolution. The system described should have particular application to the analysis of RNA viruses with multiple genome segments, and in epidemiologic studies concerned with the analysis of multiple virus isolates.

Animals↗
PubMed1990

Primer extension dideoxy chain termination nucleotide sequencing of partially purified RNA virus genomes: a technique for investigating low titre viruses with extensive genome secondary structure.

A modified version of the primer extension dideoxy chain termination nucleotide sequencing technique (Sanger et al., 1977) is described. This method has advantages over existing molecular cloning and primer extension techniques in that it allows the genome of RNA viruses to be directly sequenced from partially purified RNA preparations. Thus, viruses growing at unacceptably low titres in tissue culture can now be partially purified from infected mouse brain and sequenced. The technique also incorporates steps for the denaturation of secondary structure which has previously provided difficulties for primer extension sequencing.

Animals↗
PubMed1993

Oligonucleotide probes in infectious bronchitis virus diagnosis and strain identification.

Genomic RNA fingerprints of infectious bronchitis virus (IBV) strains M41 and Conn46 were prepared to identify T1 RNase-resistant oligonucleotides 'unique' to each of the two IBV strains. Such oligonucleotides were subsequently eluted from the gels and their nucleotide sequences determined. When oligonucleotide probes of those sequences were synthesized and used in a dot-blot hybridization assay, the probes lacked IBV strain-specificity and reacted with the RNAs of homologous as well as heterologous IBV strains. Based on these results, the methods used in this study need to be applied to a large number of oligonucleotide probes, to find one or a few that might be suitable as IBV strain- or serotype-specific oligonucleotide probes.

Animals↗
PubMed1989

Transcripts from the co-transposed segment of variant surface glycoprotein genes are in Trypanosoma brucei polyribosomes.

In Trypanosoma brucei the 5' proximal flanking sequences of a variant surface glycoprotein (VSG) gene, the co-transposed segment, are transcribed in a variant antigenic type- and stage-specific fashion along with the VSG gene. The precursor transcripts are subsequently processed to yield smaller transcripts from the co-transposed segment as well as the VSG mRNA. These co-transposed segment transcripts are quite abundant, polyadenylated and contain the spliced leader sequence, all characteristics of trypanosome mRNAs. We have found that all of the co-transposed segment transcripts from two VSG genes are present in polyribosomes. The nucleotide sequence of much of the co-transposed segment of one of these VSG genes, however, has no open reading frames coding for proteins longer than 49 amino acids. These results suggest that co-transposed segment transcripts do not encode essential proteins even though they are present in polyribosomes and may be translated.

Animals↗
PubMed1991

A novel protein antigen of the malaria parasite Plasmodium falciparum, located on the surface of gametes and sporozoites.

A Plasmodium falciparum cDNA clone was isolated of which the insert is transcribed at high rates as a 1.4-kb mRNA in the sexual stages of the malaria parasite. The cDNA clone contains a copy of a non-interrupted gene which codes for a protein of 157 amino acids (Mr = 16607). This 16-kDa protein does not contain repetitive sequences and is characterised by a putative N-terminal signal sequence, a hydrophobic membrane anchor sequence and a highly hydrophilic C-terminal region suggesting that it is an integral membrane protein. Rabbit antisera raised against a synthetic peptide covering amino acids 31-47 of the 16-kDa protein and against recombinant fusion proteins recognised the 16-kDa antigen in protein extracts of gametocytes, macrogamete/zygotes and sporozoites by Western blot analysis. The rabbit antisera also reacted with gametes, gametocytes and sporozoites in a standard immunofluorescence assay. By immunoelectron microscopy using the protein A-gold method the 16-kDa protein could be clearly visualised on the surface of macrogametes and sporozoites, whereas the antigen was not detectable in the asexual erythrocytic stages of the parasite. The 16-kDa antigen of P. falciparum therefore might have the potential to elicit a dual protective immune response against the sporozoite and sexual stage parasites.

Amino Acid Sequence↗
PubMed1989

Expression of cloned Saccharomyces diastaticus glucoamylase under natural and inducible promoters.

Any one of three homologous genes - STA1, STA2 and STA3 - encoding glucoamylase isozymes I, II and III respectively, allows the Saccharomyces species to utilize starch as a sole carbon source. We show in this paper that glucoamylase II production can be increased 4-fold over the level produced by STA2 strains, by using a two-step fermentation and a yeast strain transformed with a high-copy-number plasmid carrying the STA2 gene. The accumulation of anomalous STA2 mRNA species, mainly differing at their 5' ends, and saturation of step(s) in the secretory pathway appear to be among the major factors limiting glucoamylase expression in synthetic media.

Cloning, Molecular↗
PubMed1987

Escherichia coli RNA polymerase binding to a DNA terminus prevents formation of a closed promoter complex.

A 302 bp DNA fragment and a 113 bp subfragment of the former, both containing the fd gene VIII promoter (P VIII), were found to exhibit temperature-dependent differential behaviour in RNA chain initiation from P VIII. At 37 degrees C no significant differences were observed, while at 17 degrees C chain initiation was strongly suppressed only with the 113 bp fragment. This phenomenon depended on the presence of the (blunt) DNA terminus upstream from P VIII (position -70). Footprinting revealed that at 17 degrees C RNA polymerase was bound to this DNA fragment in a different mode. Contacts were observed only upstream from position -25. On the contrary, at 37 degrees C only the promoter complex footprint was visible. These results indicate that at 17 degrees C formation of the non-initiating complex is more favourable than formation of the promoter complex (which is closed at 17 degrees C; Hofer, B., Müller, D. and Köster, H. (1985) Nucleic Acids Res. 13, 5995-6013) and that formation of both complexes is mutually exclusive. No footprints of RNA polymerase were observed at other DNA termini. This indicates a sequence-specificity for the interaction at the terminus of the 113 bp fragment. The footprint pattern, together with features of the DNA sequence, suggests that the contacts involved in this interaction are similar to those promoter contacts formed upstream from position -20 and that DNA without a -10 region can be specifically recognized by RNA polymerase.

Bacterial Proteins↗
PubMed1990

Splice junction mutations in a yeast tRNA gene which alter the rate and precision of processing.

We have introduced mutations into a tRNALeu3 gene which alter the intron boundaries and examined their effects on RNA splicing. Our results show that the 5'-proximal splice junction is not specified by the position of an adjacent base-paired stem present in all naturally occurring tRNA precursors. Also, efficient cleavage of 5'-splice junctions unique to these mutants, -CpU-, -UpA- and -UpG-, indicates the purine found at the 5'-side of this site in all natural precursors is dispensable. Some alterations of the sequence and structure at the 5'-proximal splice site reduce the rate of cleavage therein and result in accumulation of molecules composed of the 5'-half of the tRNA plus the intron. The precise position of the 5'-proximal cleavage site can vary +/- 1 base in these mutants. The 3'-proximal splice junction is rendered inactive by changing the prospective splice junction sequence from -ApC- to -CpC- and reducing the size of an unpaired loop at this site from six to two bases. Very small amounts of RNA composed of the 3'-half of the tRNA plus the intron accumulate from this precursor. We conclude that splice junction sequence and structure affect both the rate and precision of intervening sequence removal.

Base Sequence↗
PubMed1990

Functional analysis of multiple promoters of the rat insulin-like factor II gene.

We have characterized the multiple promoters of the rat insulin-like growth factor II (rIGFII) gene by in vivo transient expression assay using a series of deletion mutant templates. Among the four promoters (P1, P2, P3 and P6), two (P2 and P3) showed relatively strong promoter activities compared with the other two. One of the four promoters, P2, was further characterized by gel band-shift and footprinting analysis using HeLa cell nuclear extract, showing two retarded bands and at least one protected sequence stretch. The results indicated that P2 has a very simple structure like P3, and consists of no more than 141 base-pairs (bp) including a TATA box and two GC core hexanucleotides. Promoter strength shown by in vivo transient expression in different cell types failed to explain the differential employment of P2 and P3 in these cells, suggesting the involvement of other regulatory mechanisms that might operate only in the native state.

Animals↗
PubMed1991

Genetic variation and evolutionary relationships amongst bluetongue viruses endemic in the United States.

The genetic variation and evolutionary relationships amongst the five serotypes of bluetongue virus (BTV) endemic to the United States were investigated by oligonucleotide fingerprint analysis. The viruses analyzed include prototype viruses of the five U.S. serotypes, and 32 viruses isolated from domestic and wild ruminants from the U.S. in the years 1979-1981. With the exception of serotype 2, most genes encoding the viral core and non-structural proteins were demonstrated to be highly conserved both within and between serotypes and some also appear to have reassorted in nature. Gene segments 2 and 6, which encode the outer capsid proteins VP2 and VP5 respectively, were more variable and were not consistently linked as serotype determination was dependent solely on gene segment 2. Gene segment 2 was the most variable gene between serotypes, but it was highly conserved within serotypes and stable over time. This suggests that the emergence of new BTV serotypes, which would require the stable incorporation of numerous mutations, must be a very slow process. Fingerprint comparisons further suggested that BTV serotypes 10, 11, 13 and 17 have evolved together in the U.S. over a considerable period of time, whereas serotype 2, which is genetically distinct, has evolved elsewhere and is most likely a recent introduction to North America.

Animals↗
PubMed1992

Analysis of relatedness of subgroup A respiratory syncytial viruses isolated worldwide.

Respiratory syncytial virus strains (subgroup A) isolated from around the world during the period 1988-1991 were analysed to determine their relatedness. Analysis was by restriction mapping and nucleotide sequencing following amplification of selected regions of the virus genome by polymerase chain reaction (PCR). Twenty-three viruses of subgroup A isolated from cities in temperate regions of the Northern and Southern hemispheres and the tropics during the period 1988-1991 fell into distinct groupings closely related to four of the six lineages defined in analysis of recurrent epidemics within the same city (Birmingham, UK) during the same period. These observations confirm that multiple lineages of RS virus co-circulate locally, and show that very similar viruses are present simultaneously in widely separated countries.

Amino Acid Sequence↗