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Biomedical subjects

R Lathe

Publications and source records attributed to R Lathe.

72 records · Page 4Linked to original sources
PubMed1984

Linker tailing: unphosphorylated linker oligonucleotides for joining DNA termini.

Current procedures for inserting linker oligonucleotides between DNA termini involve terminal addition of linker duplexes followed by restriction enzyme cleavage and religation . Such methods have the disadvantage that target DNA molecules containing internal recognition sites are cut during the linker cleavage step. We describe a method for adding linker oligonucleotides to DNA termini which eliminates the requirement for subsequent restriction enzyme treatment. Nonphosphorylated linker duplexes are ligated to target DNA termini on one strand alone. Removal of the unligated strand generates a single-stranded protrusion at each terminus. Two such tailed termini may now be linked by annealing their single-stranded complementary tails, resulting in a hybrid DNA molecule containing a single linker insertion. Each step of the procedure has been studied to optimize the recovery of recombinant molecules, and we present examples of how the method may be applied.

Cloning, Molecular↗
PubMed1983

New versatile cloning and sequencing vectors based on bacteriophage M13.

A new pair of cloning and sequencing vectors based on bacteriophage M13mp7 has been developed. These vectors (M13tg130 and M13tg131) contain, in addition to the EcoRI, BamHI, HindIII, SmaI, SalI and PstI sites present in other vectors [cf., M13mp8 and M13mp9, Messing and Vieira, Gene 19 (1982) 269-276], unique restriction recognition sequences for the enzymes EcoRV, KpnI, SphI, SstI and XbaI. A restriction site for the enzyme BglII has been incorporated into the polylinker region of one of the vector pair to permit rapid discrimination between the two vectors.

Cloning, Molecular↗
PubMed1982

Fusion of restriction termini using synthetic adaptor oligonucleotides.

A method is proposed for linking a DNA fragment possessing a 5' single-stranded extension to one carrying a 3' extension. Synthetic oligonucleotide adaptors can be used to (i) change the site specificity at the termini of a fragment generated by restriction enzyme cleavage and (ii) simultaneously dephosphorylate the extremities of a DNA molecule to prevent recircularisation and allow positive selection for recombinant DNA molecules.

Amino Acid Sequence↗
PubMed1980

Prokaryotic histone-like protein interacting with RNA polymerase.

firA mutation of Escherichia coli can render RNA synthesis thermosensitive and confer abnormal sensitivity to rifampicin, an antibiotic that specifically inhibits the activity of RNA polymerase. We previously described the cloning of a chromosomal HindIII fragment containing the firA gene, and we now present strong evidence that the product of this gene is a 17,000-dalton polypeptide which, by various criteria, closely resembles the eukaryotic histones. This protein forms the largest of a unique set of three abundant histone-like proteins (HLP) found in E. coli and is hence referred to as HLPI. We discuss possible routes by which these proteins might affect transcription.

Bacteriophage lambda↗
PubMed1977

The firA gene, a locus involved in the expression of rifampicin resistance in Escherichia coli. I. Characterisation of lambdafirA transducing phages constructed in vitro.

The firA200 mutation of E. coli not only renders RNA synthesis thermosensitive but also eliminates the high-level resistance to rifampicin associated with certain mutations in the beta subunit of the RNA polymerase. A priori, the firA gene is likely to code for an essential component of the transcription apparatus. The isolation is reported of transducing phages for the firA gene, constructed in vitro by fusing fragments of the E. coli chromosomes into a lambdoid bacteriophage. Such phages carry at least two essential genes and are able to suppress both the thermosensitivity and abnormal rifampicin sensitivity associated with the firA200 allele. The finding that some, but not all, of the lambdafirA phages have a temperature dependent growth defect is discussed.

Coliphages↗
PubMed1977

The firA gene, a locus involved in the expression of rifampicin resistance in Escherichia coli. II. Characterisation of bacterial proteins coded by lambdafirA transducing phages.

The firA gene probably codes for an essential component of the transcription machinery in E. coli. Bacterial proteins coded by lambdafirA transducing phages have been examined after infection of a UV-irradiated lambda lysogen, and 2 major fir-specific proteins have been characterized. The larger protein has a molecular weight of 27,000 daltons. The smaller protein, of 17,000 daltons, is produced in a considerable molar excess over the larger protein, is basic and binds strongly to DNA-, to DEAE- and to phospho-cellulose. This protein is clearly visible upon 2-dimensional gel electrophoresis of unfractionated E. coli protein, showing that it is present in the cell in large quantities. Evidence is presented to suggest that this protein may be identical to the Kappa factor of Schäfer and Zillig (1973).

Bacterial Proteins↗
PubMed1977

Fine-structure mapping of the firA gene, a locus involved in the phenotypic expression of rifampin resistance in Escherichia.

The firA (Ts)200 mutation not only eliminates the resistance to rifampin of certain genetically resistant strains, but, moreover, renders ribonucleic acid synthesis thermolabile. The firA gene has been mapped by P1 tranduction and is located extremely close to the structural gene for deoxyribonucleic acid polymerase III at 4 min on the Escherichia coli linkage map.

Chromosome Mapping↗
PubMedDate not supplied

Expression of rabies virus glycoprotein from a recombinant vaccinia virus.

Rabies is one of the oldest diseases know to man, but its successful control has remained elusive. Although effective vaccines of tissue culture origin against rabies do exist, such preparations are expensive. Live vaccinia virus (VV) recombinants expressing influenza or hepatitis B antigens have recently been used to immunize against these diseases. We have now used this approach to produce a novel rabies vaccine. We first altered the rabies glycoprotein cDNA by site-directed mutagenesis and removed the poly(dG) tail. We then aligned the modified cDNA with an early VV promoter sequence inserted within a cloned copy of the vaccinia thymidine kinase gene and transfected this plasmid into VV-infected cells. Recombination between the virus and the plasmid resulted in a recombinant virus harbouring the rabies glycoprotein cDNA. Inoculation of rabbits with the live recombinant virus induced high titres of rabies virus-neutralizing antibodies, and scarification with the recombinant VV protected mice against challenge with street rabies virus.

Amino Acid Sequence↗
PubMedDate not supplied

Oral vaccination of the fox against rabies using a live recombinant vaccinia virus.

Rabies, a viral disease affecting all warm-blooded animals, is prevalent in most parts of the world, where it propagates amongst wild animals, particularly the fox and dog. The public health and economic consequences of infection in man and livestock are well known. Attempts to control the disease by vaccinating wild carnivores with inactivated or attenuated rabies virus remain controversial, and we have instead evaluated here the potential of a recombinant vaccinia virus to protect foxes against the disease. We have found that the administration of vaccinia virus (VV) or a recombinant harbouring the rabies surface antigen gene (VVTGgRAB) is innocuous to foxes. The recombinant virus can elicit the production of titers of rabies-neutralizing antibodies equal or superior to those obtained with conventional vaccine, and 10(8) plaque-forming units (PFU) of VVTGgRAB administered subcutaneously, intradermally or orally confers complete protection to severe challenge infection with street rabies virus.

Administration, Oral↗
PubMedDate not supplied

Tumour prevention and rejection with recombinant vaccinia.

Tumour-specific antigens (TSA; ref. 1) have been exploited in the diagnosis and imaging of human cancer and anti-TSA antibodies have therapeutic potential. Vaccination with TSA or anti-idiotypic (TSA) antibodies has also been used to control tumour growth in model systems. An effective immune response nevertheless demands copresentation of antigen with host histocompatibility determinants. We therefore examined whether live vaccinia virus recombinants expressing TSA in cells of the vaccinated host might better elicit tumour immunity. Polyoma virus (PY) is tumorigenic in rodents; because killed PY-transformed cells can elicit tumour immunity, a PY-specific TSA has been postulated. Tumorigenesis involves expression of three early PY proteins, large-T (LT), middle-T (MT) and small-T (ST), but their role as TSAs is unclear. We therefore expressed the three T proteins in separate vaccinia recombinants. Rejection of PY tumours was observed in rats immunized with recombinants expressing either LT or MT. Further, tumour-bearing animals could be induced to reject their tumours by inoculation of recombinants.

Animals↗
PubMedDate not supplied

Development of animal recombinant DNA vaccine and its efficacy in foxes.

Rabies is prevalent in most parts of the world. An extensive reservoir of the disease is present in the population of wild animals. The fox in particular is a major vector of the disease in North America and Europe. Although attempts to control rabies by vaccination of wild carnivores with attenuated rabies virus have met with some success, this approach remains controversial. The potential of a recombinant vaccinia virus expressing the rabies glycoprotein for the protection of foxes against rabies was examined. Both the parental (wild-type) and recombinant viruses were found to be innocuous to foxes. Inoculation of live recombinant virus via the subcutaneous, intradermal, or oral routes uniformly elicited high titers of neutralizing antibodies, and animals that received 10(8) plaque-forming units of the recombinant virus in bait resisted severe challenge with live rabies virus.

Animals↗
PubMedDate not supplied

Influence of dehydroepiandrosterone on rabbit intraocular pressure.

PURPOSE: The systemic concentration of dehydroepiandrosterone decreases with age in primates while in humans intraocular pressure (IOP) increases with aging. This study was designed to investigate if a relationship existed between dehydroepiandrosterone and IOP in pigmented rabbits. METHODS: Animals were treated unilaterally for 6 weeks with topical 3% dehydroepiandrosterone in 30% 2-hydroxypropyl-beta-cyclodextrin; the contralateral eye received vehicle alone. Drops were applied, and IOP measured, twice daily. RESULTS: Small, but statistically significant, drug-related effects were found. IOP was consistently higher in the afternoon; the afternoon minus morning difference in IOP, however, decreased with time. Topical, radioactive drug application indicated very low level penetration into aqueous humor, iris, corneal epithelium, the rest of the cornea, or bulbar conjunctiva. CONCLUSION: The small drug-related effects may be due, in large part, to poor intraocular drug penetration. The circadian rhythm of IOP appears to be time-dependent in chronic studies with a gradual loss of IOP difference between a.m. and p.m. readings.

Adjuvants, Immunologic↗