Biomedical subjects
Publications and source records attributed to M Zasloff.
Pseudomonas aeruginosa bacteriophages were recovered from the sputum of patients with chronic P. aeruginosa bronchopulmonary infection. Host ranges of some of the phages are presented.
About 12 tRNAimet genes have been found at scattered locations in the human genome. Four fragments of human fetal liver DNA ranging in size from 11 to 18 kb, each containing a single tRNAimet gene, were cloned from a recombinant phage library. On the basis of restriction site mapping, electron microscopic analysis of heteroduplex structures and the maps of sequences transcribed in vivo in human fibroblasts, obtained by a novel contract-hybridization method, the fragments were shown to represent two different loci with homologies limited to several dispersed repetitive sequences within each of the chromosomal neighborhoods. Detailed structural analysis of the tRNA regions revealed several blocks of homology at the proximal flanking sequences of the two nonallelic genes, one of which differed from the common vertebrate tRNAimet sequence by a base substitution at position 56 with a T in place of G. Two oligonucleotides identical or very similar to tRNAimet structural sequences were present at the 5' border of both genes. A review of published sequence data showed other unequivocal examples of tRNA-coding sequences present at the 5' flanking region of the associated tRNA gene, which in several cases contained the site of transcription initiation.
The organization of repetitive sequences within three cloned chromosomal segments from human fetal liver DNA containing the initiator methionine tRNA gene was studied. The procedure developed for this study involves the contact hybridization of an electrophoretically separated 5'-32P-labeled restriction endonuclease digest of the cloned segment with total human genomic DNA covalently bound to aminobenzyloxymethyl-paper. The extent of hybridization of each labeled fragment to the area of paper with which it is in contact is proportional to the representation of the sequence within the human genome. We show that sequences with a wide range of genomic repetitition are present in the neighborhoods of the three dispersed initiator tRNA loci, each characterized by a different overall organization pattern.
We have extended the use of pancreatic DNase I as a probe of chromatin structure by exploring the accessibility of an active gene to the introduction of the first single-stranded nick. We show by a target analysis that the beta-globin gene is about 25-fold more sensitive to single-site nicking than is an average sequence in the chicken erythrocyte nucleus or the nontranscribed albumin gene. The sites of initial DNase I nicking are shown to cluster within the transcribed sequence of the beta-globin gene.
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A new technique has been developed for the rapid isolation of covalently closed circular DNA molecules. The procedure is a selective extraction based on differences in the partitioning of covalently closed circular DNA molecules and noncovalently closed species between phenol and water at acid pH and low ionic strength. Under the conditions described, linear as well as nicked circular DNA is extracted into phenol, while covalently closed circular DNA molecules remain in the water phase. The method permits the quantitative isolation of covalently closed circular DNA from either total cellular DNA or partially purified preparations, to a degree of purity comparable with buoyant density procedures.
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We have employed mercury-substituted UTP to study the transcription of duck reticulocyte chromatin in vitro by Escherichia coli RNA polymerase. We find that the use of this method results in large overestimates of the amount of de novo synthesis of globin-specific RNA sequences. The artefact arises because endogenous globin RNA can serve as a template for the RNA polymerase, resulting in the formation of a duplex product in which one strand is the endogenous message, and the other is the mercury-labeled complementary strand. Subsequent purification of the mercury-substituted RNA on thiol-agarose results in copurification of endogenous globin sequences. We document the details of this mechanism and describe methods which will eliminate the artefact.
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Eukaryotic cells contain polypeptide chain initiation factors that, like the prokaryotic initiation factor IF-2, promote the AUG-dependent binding of fMet-tRNA(f) to the small ribosomal subunit. The bound amino-acyl-tRNA is directly convertible to fMet-puromycin upon addition of 60S subunit. The reaction is sensitive to initiation inhibitors such as aurintricarboxylic acid and edeine but, unlike its prokaryotic counterpart, it does not require GTP. Factors that catalyze the binding and fMet-puromycin reactions with ribosomal subunits from Artemia salina embryos are present in postribosomal supernatants of Artemia, mouse fibroblasts (L cells), and rat liver, as well as in salt washes of rabbit reticulocyte ribosomes. However, whereas all three supernatant factors, like Escherichia coli IF-2, are sensitive to SH-binding reagents, the reticulocyte factor is not. The rat liver and Artemia factors function indiscriminately with Artemia or rat liver ribosomes, but the Artemia factor and E. coli initiation factor IF-2 are not interchangeable.
The supernatant initiation factor from Artemia salina embryos promotes, besides the AUG-dependent binding of fMet-tRNA(f), the poly(U)-dependent binding of N-acetylPhe-tRNA to 40S ribosomal subunits; the bound N-acylaminoacyl-tRNA reacts directly with puromycin upon addition of 60S subunits. Both the binding reaction and the synthesis of N-acylaminoacyl-puromycin occur in the absence of GTP or other ribonucleoside triphosphates. To a smaller extent, the factor also mediates the 40S ribosomal binding of Met-tRNA(f) and Phe-tRNA; in this case, the bound aminoacyl-tRNA is less reactive with puromycin. After the poly(U)- and supernatant factor-dependent binding of N-acetylPhe-tRNA to 40S subunits at low Mg(2+) concentration, binding of a second aminoacyl-tRNA (Phe-tRNA), with ensuing formation of the first peptide bond, is dependent upon the addition of the 60S subunit, elongation factor EF-1, and GTP. Further growth of the polypeptide chain requires translocation and is, therefore, dependent upon the addition of elongation factor EF-2. As with the Escherichia coli system, once requirements for translation of the third codon have been met, no further additions are necessary for elongation of a peptide chain.
Embryos of the brine shrimp, Artemia salina, were used in a study of polypeptide chain initiation in an in vitro system from a eukaryote. A protein, isolated from the high-speed supernatant, has been highly purified and shown to have properties that suggest it is the eukaryotic equivalent of the Escherichia coli initiation factor F(2): It promotes the AUG-dependent binding of fMet-tRNA (E. coli) to the Artemia 40S ribosomal subunit, but not to either the 60S or 80S species; the bound fMet-tRNA is placed in a site on the smaller subunit from which it reacts with puromycin upon addition of the 60S subunit; and the activity is sensitive to aurintricarboxylic acid and edeine, specific inhibitors of initiation. The factor, a basic protein of molecular weight about 100,000, is inactivated by N-ethylmaleimide, an SH-binding reagent, and is clearly distinct from the Artemia elongation factors, T(1) and T(2). In addition, the factor stimulates the poly(U)-dependent binding of Phe-tRNA (E. coli) to the Artemia 40S ribosomal subunit. This reaction, though similar to the fMet-tRNA-binding reaction, differs in that the bound Phe-tRNA is largely resistant to release by puromycin.
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The Alu sequence family comprises the major dispersed repeat sequences of rodent and primate genomes, numbering greater than 300,000 copies in the human haploid genome. The function of these elements is unknown. The sequences can be transcribed by RNA polymerase III and represent a substantial fraction of total heterogeneous nuclear RNA. Alu sequences can be found both in the flanking regions and within the transcription units of several well-characterized genes. Here we show that some members of the mouse B1 Alu sequence family encode a small cytoplasmic RNA. The mouse B1 sequence is congruent to 130 nucleotides long and shows homology with the monomeric units of the dimeric 300-nucleotide primate sequence. By means of microinjection studies in the Xenopus laevis oocyte, we have elucidated a novel pathway leading to the appearance of a processed B1-type Alu RNA species in the cytoplasm. The abundance of this small Alu RNA differs between various mouse tissues, suggesting a role in tissue-specific gene expression.