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T Blumenthal

Publications and source records attributed to T Blumenthal.

At least 55 records · Page 3Linked to original sources
PubMed1988

The C. elegans trans-spliced leader RNA is bound to Sm and has a trimethylguanosine cap.

mRNA splicing in C. elegans is unusual: most introns are very short (approximately 50 bases), and many mRNAs receive a leader by trans-splicing. The donor in trans-splicing is a 94 nucleotide molecule, termed the leader RNA, that contributes its 5' 22 nucleotides to a variety of mRNAs. We show here that C. elegans has the usual snRNAs, which presumably catalyze the splicing reactions. As expected, they are bound to the Sm antigen and have 2,2,7-methylguanosine caps. Remarkably, the trans-spliced leader RNA is also Sm-associated and has this special cap. Hence, a molecule discovered as a substate of splicing has properties of molecules heretofore known only to facilitate splicing of other RNAs. Mature mRNAs that have received the leader evidently lack 2,2,7-methylguanosine caps, suggesting that these caps are removed or altered during processing.

Animals↗
PubMed1988

Regulated expression of a vitellogenin fusion gene in transgenic nematodes.

In Caenorhabditis elegans the vitellogenin genes are expressed abundantly in the adult hermaphrodite intestine, but are otherwise silent. In order to begin to understand the mechanisms by which this developmental regulation occurs, we used the transformation procedure developed for C. elegans by A. Fire (EMBO. J., 1986, 5, 2673-2680) to obtain regulated expression of an introduced vitellogenin fusion gene. A plasmid with vit-2 upstream and coding sequences fused to coding and downstream sequences of vit-6 was injected into oocytes and stable transgenic strains were selected. We obtained seven independent strains, in which the plasmid DNA is integrated at a low copy number. All strains synthesize substantial amounts of a novel vitellogenin-like polypeptide of 155 kDa that accumulates in the intestine and pseudocoelom, but is not transported efficiently into oocytes. In two strains examined in detail the fusion gene is expressed with correct sex, tissue, and stage specificity. Thus we have demonstrated that the nematode transgenic system can give proper developmental expression of introduced genes and so can be used to identify DNA regulatory regions.

Animals↗
PubMed1987

A single gene encoding vitellogenin in the sea urchin Strongylocentrotus purpuratus: sequence at the 5' end.

The synthesis of vitellogenin (yolk protein precursor) in the sea urchin, Strongylocentrotus purpuratus, is unique in that both males and females produce a high level of the protein. In this paper we show that this organism also is unique in possessing only a single vitellogenin gene. Like the genes that encode analogous proteins in vertebrates, the sea urchin gene is large, about 19 kb in length. The sequence surrounding the 5' end of the gene revealed several other similarities to vertebrate vitellogenin genes: the signal sequence is exceptionally short and has a sequence similar to those from frog and chick; there is a canonical TATA box at -32; and there is a sequence closely resembling the estrogen-responsive element at -207.

Amino Acid Sequence↗
PubMed1986

Characterization of regions of the Caenorhabditis elegans X chromosome containing vitellogenin genes.

Caenorhabditis elegans contains a family of vitellogenin genes consisting of five closely related genes (vit-1 to vit-5) coding for 186,000 Mr yolk proteins, and one distantly related gene (vit-6) encoding a 200,000 Mr precursor to two smaller yolk proteins. We demonstrate here that, although vit-1 to vit-5 are not clustered (with the exception of vit-3 and vit-4), they are all on the X chromosome. In contrast, vit-6 is autosomal. The genes are strictly regulated during development: they are activated in the intestine of the hermaphrodite worm, following the last larval molt. In order to determine whether the vit genes are contained within chromosomal domains of similarly regulated genes, we have used the chromosomal "walking" technique to isolate 55,000 to 60,000 base-pairs of DNA surrounding each of the X-linked genes and determined the developmental specificity of nearby genes. In the total of 235,400 base-pairs of cloned DNA, seven genes, in addition to the five vit genes, were found. The average gene spacing is approximately 20,000 base-pairs per gene but is highly variable, ranging from less than 2000 to more than 38,000 base-pairs. The seven newly identified genes, called uvt-1 to uvt-7, specify RNAs varying in size from 500 to 2700 bases. With the exception of uvt-4, all of the genes are developmentally regulated; but the patterns of regulation are quite variable, and all are different from the vitellogenin genes. The vit genes, therefore, are not contained within co-regulated chromosomal domains. We also searched for the presence of repetitive DNA, but only four such sequences were found.

Animals↗
PubMed1986

Expression of the vitellogenin gene in female and male sea urchin.

Expression of vitellogenin, the yolk protein precursor, is strictly regulated during development. In previous studies on a variety of organisms, vitellogenin gene expression has been shown to be restricted to one or two tissues in adult female animals. In this report we show that, in contrast, sea urchin vitellogenin is synthesized in both females and males. To identify sea urchin vitellogenin, we raised antibodies specific for the major yolk protein. We show here that a 155-kDa polypeptide, immunoprecipitable by the antibody to the major yolk protein, is synthesized in the intestines of female and male sea urchins and also in ovaries and testes. This 155-kDa polypeptide is converted to a 195-kDa vitellogenin in each of these tissues; further modification to yield the 180-kDa major yolk protein occurs only in the ovary. We have also identified a vitellogenin cDNA clone and used it to study vitellogenin mRNA production. An abundant 5.1-kilobase mRNA was found in the tissues containing vitellogenin. Our results suggest that vitellogenin may serve the following two functions in sea urchins: its classical role as a yolk protein precursor and an unidentified function required by adults of both sexes.

Animals↗
PubMed1985

The nucleotide sequence of a nematode vitellogenin gene.

The nematode, Caenorhabditis elegans, contains a family of six genes that code for vitellogenins. Here we report the complete nucleotide sequence of one of these genes, vit-5. The gene specifies a mRNA of 4869 nucleotides, including untranslated regions of 9 bases at the 5' end and 51 bases at the 3' end. Vit-5 contains four short introns totalling 218 bp. The predicted vitellogenin, yp170A, has a molecular weight of 186,430. At its N terminus it is clearly related to the vitellogenins of vertebrates. However, the vit-5-encoded protein does not contain a serine-rich sequence related to the vertebrate vitellin, phosvitin. In fact, the amino acid composition of the nematode protein is very similar to that of the vertebrate protein without phosvitin. Vit-5 has a highly asymmetric codon choice dictionary. The favored codons are different from those favored in other organisms, but are characteristic of highly expressed C. elegans genes. The strong selection against rare codons is not as great near the 5' end of the gene; rare codons are 15 times more frequent within the first 54 bp than in the next 4.8 kb.

Amino Acid Sequence↗
PubMed1985

The C. elegans vitellogenin genes: short sequence repeats in the promoter regions and homology to the vertebrate genes.

The nematode Caenorhabditis elegans contains a small family of vitellogenin genes which is expressed abundantly, but only in the intestine of the adult hermaphrodite worm. In order to identify possible regulatory elements, we have sequenced the DNA surrounding the 5' ends of five of the six genes. Contained within regions which have largely diverged from one another, two different heptameric sequences are found repeated within the first 200 bp upstream of each of the genes. The first sequence, TGTCAAT, is present as a perfect heptamer at least once upstream of each gene. It is repeated in both orientations four to six times in each 5' flanking region, allowing a one-base mismatch. The second sequence, CTGATAA, is also present as a perfect heptamer in a restricted region upstream of each gene. These two sequence elements may be involved in regulation of the vitellogenin genes. Remarkably, the CTGATAA sequence is present in a similar location in the promoter regions of vertebrate vitellogenin genes. In fact, our data reveal a surprising degree of similarity between the nematode and vertebrate vitellogenins.

Amino Acid Sequence↗
PubMed1985

The Caenorhabditis elegans vitellogenin gene family includes a gene encoding a distantly related protein.

While the nematode Caenorhabditis elegans is more primitive than most egg-laying organisms, it's vitellogenins, or yolk protein precursors, appear to be more complex. C. elegans oocytes accumulate two major classes of yolk proteins. The first consists of two polypeptides with an Mr of about 170,000 (yp170A and yp170B) encoded by a family of five closely related genes called vit-1 through vit-5. The second class consists of two smaller proteins with Mr values of 115,000 (yp115) and 88,000 (yp88) which are cut from a single precursor. Here we report the cloning and analysis of a single-copy gene (vit-6) that encodes this precursor. The lengths of the gene and its mRNA are about 5 X 10(3) base pairs. Like vit-1 through vit-5, vit-6 is expressed exclusively in adult hermaphrodites. Comparison of portions of the coding sequence indicates that vit-6 is distantly related to the vit-1 through vit-5 gene family. Thus, even though the two classes of yolk proteins are antigenically and physically distinct, they are encoded by a single highly diverged gene family.

Animals↗
PubMed1984

Cloning of a yolk protein gene family from Caenorhabditis elegans.

We have cloned a family of five genes which encode the 170,000 Mr yolk proteins in the nematode Caenorhabditis elegans. The genes and their messenger RNAs are about 5 X 10(3) base-pairs in length. Thus most of the length of each gene is exon, although a few small introns have been discovered. Based on hybridization and restriction mapping experiments, the genes can be subdivided into two subfamilies: YP1-YP2 and YP3-YP4-YP5. Within a subfamily the genes are nearly identical. While most of the genes are not clustered, YP3 and YP4 are tandemly linked. Hybrid-arrest translation experiments demonstrate that the YP3-YP4-YP5 subfamily encodes the yp170A yolk protein, while the YP1-YP2 subfamily encodes the yp170B yolk protein. RNAs homologous to these genes are abundant in the adult hermaphrodite, but missing from larvae and males. Furthermore, RNA isolated from dissected intestines is highly enriched for sequences that hybridize to the genes, whereas RNA from gonad or body wall is nearly devoid of these sequences. Thus, this gene family is apparently expressed only in the intestine of the adult hermaphrodite.

Animals↗
PubMed1983

Does Q beta replicase synthesize RNA in the absence of template?

Q beta replicase, in the absence of added template, will synthesize RNA autocatalytically. A variety of small RNa species, termed '6S RNAs' are generated. As this reaction purportedly occurs in the absence of template, it has been termed 'de novo' RNA synthesis. The question of whether Q beta replicase can polymerize replicatable RNA molecules, without instruction from a template, has important evolutionary implications. The finding that Q beta replicase was able to synthesize RNA de novo was based on (1) failure to find contaminating RNA in Q beta replicase preparations; (2) differences in the sizes of products of apparently identical reactions; and (3) kinetic differences between template-instructed and de novo reactions. Here wer describe a procedure for production of Q beta replicase lacking one of its subunits, ribosomal protein S1, involving column chromatography in the presence of a low concentration of urea. We show that the resulting highly purified enzyme will not synthesize detectable RNA in the absence of added template. We show also that the ability to perform a reaction kinetically indistinguishable from the de novo synthesis reaction can be restored to the highly purified enzyme by adding a heat-stable, alkali-labile component of Q beta replicase preparations. Thus our findings suggest that, in the novo reaction, Q beta replicase is replicating previously undetected contaminating RNA molecules.

Chromatography, DEAE-Cellulose↗
PubMed1983

Q beta replicase containing a Bacillus stearothermophilus elongation factor.

We purified Q beta replicase containing EF-Ts from Bacillus stearothermophilus in place of the homologous polypeptide from Escherichia coli. The hybrid enzyme was fully active in the transcription of a variety of templates. It was found to be qualitatively similar to native Q beta replicase with respect to a variety of parameters which measure the efficiency of initiation of RNA synthesis. The results demonstrated that Q beta replicase can tolerate substantial alterations in the EF-Tu X Ts component of the enzyme. These alterations resulted in only minor perturbations of catalytic properties.

Escherichia coli↗
PubMed1982

Analysis of RNA polymerase by trypsin cleavage. Different structural changes produced by heparin and DNA.

Alterations in RNA polymerase structure can be detected using initial trypsin cleavage rates as a conformational probe. Both template (poly[d(A-T) . d(A-T)] and the RNA polymerase inhibitor, heparin, alter the rates at which the subunits of the enzyme are cleaved. However, while the presence of poly[d(A-T) . d(A-T)] slows the cleavage of subunits beta, sigma, and alpha by trypsin, heparin accelerates the cleavage of beta and sigma. Furthermore, the presence of heparin does not prevent the effect of poly[d(A-T) . d(A-T)] on the beta and sigma cleavage rates. Thus, heparin does not eliminate the interaction between DNA and RNA polymerase. That heparin does alter the nature of this interaction is demonstrated by the fact that template decreases the trypsin cleavage rate of subunit alpha in the absence, but not in the presence, of heparin. Like heparin, the addition of RNA to the reaction increases the accessibility of beta and sigma to trypsin. Hence the interaction of heparin with RNA polymerase may mimic the product, rather than the template, interaction.

DNA-Directed RNA Polymerases↗
PubMed1982

An interaction between gramicidin and the sigma subunit of RNA polymerase.

Gramicidin, a peptide antibiotic produced by Bacillus brevis, inhibits initiation of transcription by RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). We show here that the presence of gramicidin causes an increase in the rate of cleavage of the sigma subunit of Escherichia coli RNA polymerase by trypsin, although it does not alter the cleavage rate of any of the core subunits. Furthermore, whereas isolated sigma is cleaved much faster than is sigma in holoenzyme, gramicidin substantially decreases the trypsin cleavage rate of isolated sigma. Inhibition of RNA polymerase activity by gramicidin in consistent with a sigma-specific effect: the antibiotic is a strong inhibitor of transcription of T7 phage DNA, which requires sigma for activity, but it has little effect on transcription of sigma-independent templates, such as poly(dA-dT).poly)dA-dT) and calf thymus DNA. These results are discussed in light of the hypothesized role for gramicidin in the initiation of sporulation of B. brevis.

DNA, Viral↗
PubMed1981

Molecular properties of two mutant species of the elongation factor Tu.

The molecular properties of two mutant species of the elongation factor Tu (EF-Tu), derived from either tuf A or tuf B, have been studied. One, designated EF-TuAR, is the product of a kirromycin-resistant tufA gene. The other designated EF-TuBO is a tuf B product and is present in a kirromycin-resistant mutant of Escherichia coli (LBE 2012) also harbouring the EF-TuAR species. EF-TuAR has been isolated in homogeneous form as a single gene product from the mutant strain LBE 2045, in which the tuf B gene has been inactivated by an insertion of the bacteriophage Mu. EF-TuBO has been isolated from LBE 2012 together with EF-TuAR in a 1:1 mixture. Fractionation of this mixture of DEAE-Sephadex A-50 resulted in an enrichment of EF-TuBO of about 80%. The properties of EF-TuAR and EF-TuBO have been compared to those of a kirromycin-sensitive species designated EF-TuAS, which was isolated from LBE 2045 by transduction of wild-type tuf A. We show here that all three EF-Tu species are fully competent to sustain polypeptide synthesis. All also appear to interact normally with guanine nucleotides and EF-Ts. Only in the presence of the antibiotic do the following differences appear. (a) Kirromycin causes EF-TuAS (wild-type tuf A gene product) to be retained on, and thus block, the ribosome. (b) EF-TuAR fails to bind the antibiotic and thus is capable of protein synthesis in its presence. (c) EF-TuBO fails to sustain polypeptide synthesis upon binding of kirromycin. It does not, however, block the ribosome, so the strain harbouring both this protein and EF-TuAR (LBE 2012) is kirromycin resistant.

Anti-Bacterial Agents↗
PubMed1980

Roles of the host polypeptides in Q beta RNA replication. Host factor and ribosomal protein S1 allow initiation at reduced GTP concentration.

Initiation of transcription of favored templates by Q beta replicase requires a much lower GTP concentration than does transcription of templates that are selected against. Although the enzyme requires a high GTP concentration to initiate transcription of Q beta RNA, the presence of the host factor substantially reduces the GTP concentration requirement. In addition, the Q beta replicase preparation must contain ribosomal protein S1 for initiation to occur at low GTP. Mn2+ ions, which can substitute for host factor in Q beta RNA replication in vitro, and which reduce the template specificity of Q beta replicase, also reduce the GTP requirement for initiation. But while Mn2+ ions produce this effect with all templates, host factor is specific for Q beta RNA. When both host factor and Mn2+ are present, transcription of Q beta RNA occurs at a much lower GTP concentration. Thus, host factor and Mn2+ appear to reduce the GTP concentration requirement by different mechanisms.

Carrier Proteins↗
PubMed1980

Analysis of RNA polymerase by trypsin cleavage. Evidence for a specific association between subunits sigma and beta involved in the closed to open complex transition.

We have analyzed subunit interactions of Escherichia coli RNA polymerase by measuring the rate of cleavage of each subunit by trypsin. We have modified a standard sodium dodecyl sulfate gel technique to include sodium tetradecyl sulfate, which results in a large separation of the beta and beta' subunits. A comparison of enzyme lacking the sigma subunit (core) with holoenzyme reveals that the presence of sigma does not alter the cleavage rates of alpha or beta, but it dramatically slows the initial cleavage of beta. Simple addition of purified sigma to core results in reconstitution of holoenzyme as measured both by glycerol gradient sedimentation and protection of beta from trypsin cleavage. However, Mg2+ is required for the beta protection but not for the binding of sigma to core. Although the enzyme reconstituted in the absence of Mg2+ and lacking the sigma-beta association is similar to holoenzyme with respect to promoter binding, it catalyzes the transition from closed to open complexes much more slowly than does enzyme reconstituted in the presence of Mg2+. Thus, the specific association between beta and sigma may be involved in the DNA melting phase of the RNA polymerase-DNA interaction. We have also examined the effects of the polyanion heparin, an RNA polymerase initiation inhibitor, on the rates of trypsin cleavage of holoenzyme subunits. Again, subunits alpha and beta' were unaffected, but heparin increased the cleavage rate of beta such that the rate was indistinguishable from the beta cleavage rate of core enzyme. Since heparin did not cause the release of sigma as measured by glycerol gradient analysis, the inhibitor may simply disrupt the sigma-beta association.

DNA-Directed RNA Polymerases↗