Showing papers on "Gene expression published in 1977"

Recombinational switch for gene expression.

Abstract: Flagellar antigens are specified by two genes, H1 and H2. The expression of these genes is regulated such that only one gene activity, or phase, is expressed at a given time. Molecular cloning techniques were used to isolate the segments of Salmonella DNA which contain these genetic loci. Heteroduplex analyses revealed an anomaly in the cloned fragment, that is, and apparent inversion, which was shown to be adjacent to the H2 gene. A correlation was demonstrated between the phase state of the H2 gene and the sequence of the adjacent segment. We propose that an inversion of this region is the phase-determining event in flagellar gene expression in Salmonella.

Regulation of Early and Late Simian Virus 40 Transcription: Overproduction of Early Viral RNA in the Absence of a Functional T-Antigen

Abstract: Virus-specific RNA synthesized in monkey cells after infection by both wild-type simian virus 40 (SV40) and the early SV40 temperature-sensitive mutant tsA58 has been analyzed. The fraction of SV40-specific RNA increased throughout infection with either wild-type SV40 or with tsA58 in direct proportion to the accumulation of progeny DNA molecules, suggesting their role in the late transcriptional process. Cytoplasmic fractions from cells infected at various temperatures (31.5 to 41°C) by wild-type virus and harvested 48 h later contained 4 to 8% virus-specific RNA, of which 5 to 10% was early SV40 RNA. In contrast, though 5 to 8% of the cytoplasmic RNA from tsA 58-infected cells incubated at 31.5 to 37°C for 48 h was virus specific, the percentage of early virus-specific RNA ranged from 25 to 80% as the incubation temperature increased. In tsA58-infected cultures incubated for 48 h at 41°C (a temperature at which essentially no tsA 58 DNA synthesis occurred), only 0.4% of the cytoplasmic RNA was virus specific, but at least 90% of this RNA was early. In experiments where cells were inoculated at 32°C and shifted at 48 h postinfection to 40°C for various times, the percentage of virus-specific pulse-labeled RNA varied from 3.5 to 10.0%. Of the virus-specific RNA, early SV40 RNA ranged from 14 to 65% in tsA 58-infected cultures. Analogous studies with Sarkosyl-extracted viral transcription complexes to incorporate label into nascent (unprocessed) viral RNA yielded essentially identical results. This finding strongly suggests that the overproduction of early SV40 RNA occurs at the level of synthesis. While cytosine arabinoside effectively terminated most viral DNA replication in wild-type-infected cells, the ratio of early to late viral RNA remained less than 1:9. These results demonstrate that: (1) the amount of virus-specific RNA synthesized depends directly on the amount of viral DNA available for use as templates; once viral DNA replication has occurred, presumably providing progeny SV40 DNA molecules for templates, the level of transcription remains high; (ii) termination of viral DNA replication does not terminate late SV40 transcription; (iii) early SV40 RNA is overproduced by tsA 58 at all temperatures, but especially at higher temperatures; and (iv) overproduction of early SV40 RNA appears to be correlated with defectiveness of the tsA mutant T-antigen. These results suggest that T-antigen may regulate its own production either by repressing the synthesis of early viral RNA or by stimulating the synthesis of late SV40 RNA or both.

Interrelationships and functions of the pyruvate kinase isozymes and their variant forms: a review.

Abstract: The relationships among and the properties of the pyruvate kinase isozymes are reviewed, emphasizing their potential role in carcinogenesis. Particular consideration is given to evaluation of the concept that the three major nonreadily interconvertible forms are the products of distinct genes, the relationship of these forms to additional separable forms of pyruvate kinase, the types and possible functions of interconvertible forms of the major isozymes, and mechanisms affecting the genetic expression of the isozymes. Emphasis is placed upon the apparent derepression of the fetal isozyme in hepatomas and the influence of neoplasms and their extracts on the expression of pyruvate kinase in the liver of host animals.

Design of molecular control mechanisms and the demand for gene expression.

Abstract: Regulation by a repressor protein is the mechanism selected when, in the organism's natural environment, there is low demand for expression of the regulated structural genes. Regulation by an activator protein is selected when there is high demand for expression of the regulated structural genes. These general conclusions are useful in relating physiological function to underlying molecular determinants in a wide variety of systems that includes repressible biosynthetic pathways, inducible biosynthetic enzymes, inducible drug resistance, and prophage induction, as well as inducible catabolic pathways, for which a special case of this prediction previously was reported [Savageau, M. A. (1974) Proc. Natl. Acad. Sci. USA 71, 2453-2455].

Enzymic Differentiation of Human Liver: Comparison with the Rat Model

Abstract: Summary: The quantitative pattern of enzymes in the second trimester human fetal liver is significantly different from that of adult liver. For some 20 enzymes, the activity quotient (AQ, i.e., activity of immature liver divided by that in adult liver) is appreciably different from 1.0. Most of the enzymes increase their concentrations with age but, as one would expect, some contribute to differentiation by diminishing in amount. In developing human liver the concentrations of the various enzymes tend to change in the same direction as they do in rat liver. Those that increase in rat liver have been classified into three main clusters, according to whether their rise begins on about the 17th day of gestation (B), the first neonatal day (C), or just before weaning (D), respectively. The distribution of these enzymes among these three clusters correlates with their AQ's in the human fetal liver. In general, enzymes with AQ around 0.5 belong to cluster (B) in rat liver whereas those with 0–0.16 belong to cluster C or D. Gross malformations resulting from the teratogenic action of drugs, hormones, or vitamins on the early embryo attract much attention. The harmful impacts of such agents at late stages of gestation are less spectacular. They may be more frequent, however, and manifest themselves in permanent inadequacies in metabolism or growth with a tendency to succumb to minor childhood diseases. The underlying causes may not be mirrored in the cytocomposition or even the subcellular morphology of autopsy specimens. Only deviations from the organ characteristic quantitative pattern of gene products would provide sensitive enough indictors of the metabolic lesions and of the aberrant aspects of differentiation that were responsible for them. In both the presence and absence of detectable morphologic abnormalities, the study of enzymes, this most varied and largest class of specific chemical constituents, would greatly extend the resolving power of the usual diagnostic procedures postmortem. Speculation: The sequence in which different enzymes approach their adult concentration in human liver closely resembles that in rat liver. This suggests that the mechanisms responsible for the schedule of gene expression must also be analogous: the synthesis of specific groups of enzymes at each corresponding critical period is regulated by the same hormones in both species. Hence, the “enzyme pathology” of infant livers not only specifies basic metabolic lesions: aided by observations on the rat model, it could also identify the age of onset and the kind of aberrations in the fetal environment which initiated the lesions.

Macromolecular Synthesis in Cells Infected by Frog Virus 3. VII. Transcriptional and Post-Transcriptional Regulation of Virus Gene Expression

Abstract: We have used improved techniques for separating individual species of RNA and protein to study the mechanisms that control gene expression by frog virus 3, a eucaryotic DNA virus. Forty-seven species of viral RNA and 35 viral polypeptide species were resolved by polyacrylamide gel electrophoresis. The relative molar ratios of virus-specific polypeptides synthesized at various times after infection were determined by computer planimetry and were compared with the molar ratios of appropriate-sized viral RNAs to code for each polypeptide. Viral polypeptides were classified according to the time during the growth cycle at which their maximal rate of synthesis occurred - early, 2 to 2.5 h; intermediate, 4 to 4.5 h; and late, 6 to 6.5 h. The viral RNAs, which were assumed to be mRNA's, could not be classified according to time of maximum synthesis; once their synthesis had begun, most of the RNAs continued to be synthesized at the same or higher rates. However, only 10 of the 47 viral RNA bands were plainly visible after electrophoresis of extracts from cells labeled from 1 to 1.5 h after infection; these 10 RNAs were designated "early" RNA. The early pattern of both RNA and polypeptide synthesis was maintained for at least 6 h in the presence of the amino acid analog fluorophenylalanine, which indicates that a functional viral polypeptide was required for "late" transcription and translation. The presumptive mRNA's for late polypeptides did not appear until 2 h after infection, but two of these "late" RNAs became the major products of transcription by 4 h into the infectious cycle. In contrast to the declining rate of synthesis of the early proteins, corresponding early RNA species continued to be synthesized at the same or higher rates throughout the replicative cycle. Although the synthesis of late virus-specific proteins appeared to be regulated at the level of transcription, our results suggest that the synthesis of both early and intermediate proteins was regulated at the post-transcriptional level.

Sequence of cro gene of bacteriophage lambda

Abstract: KNOWLEDGE of the primary sequences of represser proteins and the DNA operators with which they interact is essential for detailed study of the molecular aspects of the control of gene expression. The sequences of two repressers are known: the cI protein of lambda (ref. 1 and R. Sauer and R. Anderegg, personal communication) and the i gene product of the lactose operon of Escherichia coli (refs 2,3 and P. J. Farabaugh, personal communication). Here we report the DNA sequence for the structural gene of a third represser, the Cro protein of lambda. This protein is of special interest both because of its small size (66 amino acids, as compared with the 236 amino acids of the cI protein and the 360 amino acids of the lac i gene), and because genetic evidence suggests that it interacts with the same operator regions as does the cI protein, although the two proteins probably do not recognise exactly the same DNA bases4–7.

Purification of a putative precursor of globin messenger RNA from mouse nucleated erythroid cells.

Abstract: Nucleated erythroid cells were incubated for 10 min in the presence of [5-3H]uridine, and the total RNA was isolated by three different extraction procedures. RNA containing globin messenger RNA sequences was purified from other cellular RNAs by selective hybridization to globin complementary DNA cellulose. Depending upon the extraction procedure employed, 0.4-0.6% of the radioactively-labeled total cellular RNA applied to the column annealed to globin complementary DNA cellulose. The annealed RNA was treated with formaldehyde and analyzed by formaldehyde/polyacrylamide gel electrophoresis. Mature globin mRNA and an RNA migrating at approximately 15 S were observed. No globin mRNA containing sequences larger than 20 S were present. The 15S RNA was partially resolved from mature globin mRNA by neutral sucrose density gradient centrifugation. The RNA isolated from the heavy region of this gradient migrated as 15 S in the formaldehyde/polyacrylamide gels and retained its ability to quantitatively anneal to globin complementary DNA cellulose. On the basis of these observations, we conclude that nucleated erythroid cells obtained from the spleens of anemic mice have a 15S RNA which contains globin mRNA sequences. The 15S RNA is not an aggregate and is a good candidate for a globin mRNA precursor.

A mechanism for the induction and regulation of human fibroblastoid interferon genetic expression.

Abstract: Summary Some commonly used inducers of interferon such as viruses and double-stranded RNA are known to inhibit cellular protein synthesis. We have now shown that conventional inhibitors of macromolecular synthesis such as cycloheximide, 2-(4-methyl-2, 6-dinitroanilino)-N-methyl propionamide (MDMP) and 1-β-d-ribofuranosylbenzimidazole (DRB) can induce the production of significant amounts of interferon in human fibroblastoid cell lines. The interferon-inducing activity of these inhibitors depends on the concentration as well as the time of treatment with the inhibitors. These findings lead to the suggestion that the induction of human interferon may be mediated by a reduction in the critical concentration of a rapidly turning over repressor(s) which normally represses the interferon gene(s) in uninduced cells.

Effects of glucocorticoids on casein gene expression in the rabbit.

Abstract: Milk protein synthesis is initiated by prolactin and a glucocorticoid. In the rabbit, prolactin alone is sufficient. However, glucocorticoids potentiate the action of prolactin. The stimulatory effect of glucocorticoids was evaluated after injections of hydrocortisone acetate alone or associated with prolactin by measurements of (a) the total RNA and DNA content of mammary glands, (b) the lactose synthetase activity, (c) casein synthesis, and (d) the concentration of casein mRNA in total cellular RNA and in polysomal RNA by hybridization with its cDNA. The glucocorticoid, totally inactive alone, proved to have a stimulatory effect proportional to the dose injected when prolactin was present. This effect was more evident with low doses of prolactin. Glucocorticoids proceeded by amplifying the capacity of prolactin to enhance the concentration of casein mRNA available for translation. A parallel effect of glucocorticoids on translation of casein mRNA was suspected. Glucocorticoids injected with low doses of prolactin were unable to mimic all the effects of high doses of prolactin alone.

Interaction of E. coli RNA polymerase with promotors of coliphage T5: the rates of complex formation and decay and their correlation with in vitro and in vivo transcriptional activity.

Abstract: The genome of virulent coliphage T5 contains about 30 sites which form stable complexes with E. coli RNA polymerase. Some of these sites bind RNA polymerase with high rates, others form extremely stable complexes as compared with promotors of other E. coli systems. The transcriptional activity of these promotors in vivo and in vitro reflects the rate of complex formation with RNA polymerase rather than the stability of the enzyme/promotor complex. The fastest, i.e. the most active promotors are found in the "early" region of gene expression followed by promotors of the "preearly" class. The few binding sites for the E. coli holoenzyme within the "late" region react more slowly with the enzyme.

Somatic nuclei in amphibian oocytes: evidence for selective gene expression

Abstract: Previous work has shown that multiple HeLa nuclei injected into Xenopus oocytes remain transcriptionally active for many days and that the expression of HeLa genes in oocytes can be detected by 2-D gel electrophoresis. We show here that of 25 proteins which have the electrophoretic properties of HeLa gene products, only 3 are expressed in injected oocytes. To test that these proteins are products of HeLa genes, and not products of activated oocyte genes, we have injected HeLa nuclei into enucleated oocytes. Three days later, several HeLa proteins were synthesized. The turning off of most HeLa genes in injected oocytes is apparently not at the translational level. This is indicated by the fact that adenovirus mRNA is efficiently translated when injected into Xenopus oocytes. When adenovirus-infected HeLa cell nuclei are injected into oocytes the adenovirus genes are not expressed, although some HeLa genes are expressed by the same nuclei. The same HeLa genes as are expressed or switched off in injected Xenopus oocytes are also preferentially expressed or switched off in injected oocytes of a Urodele amphibian, Pleurodeles . This suggests that conditions or molecules may exist in oocytes which selectively impose on injected nuclei a new programme of gene expression.

Defects of glycosyltransferase activities in human fibroblasts of Pk and p blood group phenotypes.

Abstract: We demonstrate that human fibroblasts of the rare Pk phenotype lack globoside, which was identified as the blood group P antigen, and that p cells possess neither globoside nor trihexosyl ceramide, which was identified as Pk antigen. Our investigations indicate also that these glycosphingolipid patterns are most likely caused by inherited preferential biosynthetic pathways in the abnormal phenotypes rather than by excess catabolism of the antigens. Evidence is presented that the fibroblasts of Pk phenotype lack beta-N-acetylgalactosaminyltransferase (globoside synthetase; UDP-N-acetylgalactosamine:trihexosylceramide beta-N-acetylgalactosaminyltransferase; EC 2.4.1.79) activity, and those of p are deficient in alpha-galactosyltransferase (trihexosylceramide synthetase; UDP galactose:lactosylceramide alpha-galactosyltransferase) and possibly also in globoside synthetase. The diminished globoside synthetase activity in p cells, however, is not caused by the defect in the gene coding for this enzyme. It appears, rather, to be caused by a failure in gene expression because one-third of Pk X p hybrids became able to express P antigenicity with a time lag of 3-4 days after cell fusion [Fellous, M., Gerbal, A., Nobillot, G. & Weils, J. (1977) Vox Sang. 32, 262-268].

RNA synthesis of vesicular stomatitis virus. VII. Complete separation of the mRNA's of vesicular stomatitis virus by duplex formation.

Abstract: Full-length virion RNA and complementary mRNA's of vesicular stomatitis virus can be annealed to each other, digested with RNases, and then separated as five unique duplex RNA molecules on polyacrylamide slab gels. Similar RNA duplexes were detected whether mRNA or virion RNA was the radioactive component and whether the mRNA was synthesized in vitro or in vivo. The sharp banding pattern of these RNA molecules was dependent on treatment with RNase T2, suggesting that removal of poly(A) is necessary. Identification of the coding region contained in each RNA duplex was based on their previous identification as single-stranded mRNA on formamide-containing, polyacrylamide gels. Because the two smallest mRNA'S had not been previously separated, their identification was based on their in vitro transcriptional gene order. In the order of increasing mobilities on the slab gels, the RNA duplexes are identified as the hybrid of the region of the genome RNA hybridized to the complementary mRNA coding for the large protein, the glycoprotein, the nucleocapsid protein, the core-associated NS protein, and the matrix protein (L,G,N,NS, and M). Several lines of evidence support the presence of undegraded complete mRNA, excluding poly(A), in these RNA duplexes. Also, the two smallest mRNA's, separated by duplex formation, were denatured, and their individual oligonucleotide fingerprints were determined. From chemical length determinations, the molecular weights of the mRNA, minus poly(A), are 2.78 X 10(5) and 2.5 X 10(5), respectively, for the mRNA's of the NS and M proteins.

Roles of the Early Genes of Bacteriophage T7 in Shutoff of Host Macromolecular Synthesis

Abstract: Through the use of phage mutants in which various combinations of the early genes are active, and in which late gene expression is blocked, we have examined the roles of each of the five early gene products of bacteriophage T7 in regulating the synthesis of host RNA and proteins. At least two independent transcriptional controls operate during bacteriophage T7 development. The product of gene 0.7, acting alone, leads to a rapid (by 5 min) shutoff of host transcription. In the absence of gene 0.7 function, and in the absence of the phage-specified RNA polymerase, a delayed shutoff of host-dependent transcription begins at approximately 15 min after infection. This secondary control element requires either a functional gene 0.3 or gene 1.1. In the absence of any early gene products, host shutoff is not observed until much later in infection (>30 min). The delayed manner in which the products of genes 0.3 and 1.1 exert their effect suggests that their mode of action is indirect. Under conditions in which the late genes are transcribed (inefficiently) by the host RNA polymerase, gene 1.1 is observed to stimulate the synthesis of lysozyme (the product of a late phage gene). In contrast, when the late genes are transcribed by the phage-specified RNA polymerase (the product of gene 1), the kinetics of synthesis of the phage RNA polymerase itself, and of lysozyme, are not affected by the deletion of genes 0.3, 0.7, 1.1, and 1.3. We conclude that under these conditions, the products of these genes are required neither for regulation of expression of the late genes nor for the shutoff of early phage gene expression.

Endogenous RD-114 virus genome expression in malignant tissues of domestic cats.

Abstract: Endogenous xenotropic cat type C virus (RD-114)- and infectious feline leukemia virus (FeLV)-specific gene expressions were measured in spontaneous sarcomas carcinomas, and nonmalignant cat tissues by molecular hybridization for virus-specific RNA and competition radio-immunoassays for the major internal protein (p30) of these two viruses. The results indicate that RD-114 gene expression in sarcomas and carcinomas at both RNA and p30 levels is significantly higher than histologically normal tissues from cats free of cancer. In contrast, the levels of FeLV viral RNA and p30 are fount to be low or undetectable in the majority of these tumored and normal tissues examined. Whereas variability in the amounts of RD-114 OR FeLV RNA and p30 expressed is found in tissues from different cats, their expression is fairly uniform in multiple malignant tissues of the same cat. The finding of widespread occurrence of elevated RD-114 gene expression in sarcomas and carcinomas is consistent with our similar observation with natural lymphomas of domestic cats and suggests that expression of certain functions of this endogenous virus may be etiologically involved in the development of many different spontaneous neoplasms of cats.

Early gene expression of adenovirus type 2: R-loop mapping of mRNA and time course of viral DNA, mRNA, and protein synthesis.

Abstract: Adenovirus type 2 DNA was hybridized to early mRNA isolated from the cytoplasm of infected cells prior to the initiation of viral DNA synthesis. Resulting R loops were visualized in the electron microscope, and their positions were oriented with the help of DNA fragments generated by digestion with the restriction endonuclease BamHI. Early RNA was found to map (in order of relative R-loop frequency) with midpoints near positions 0.95, 0.80, 0.03, 0.65, and 0.09 on the conventional adenovirus map. The time of appearance of individual viral mRNA's was compared to the time course of viral protein and DNA synthesis. We present a refined map of adenovirus gene functions which is based on results documented in this and the accompanying study by Meyer et al. (1977), as well as on data published by other laboratories.

Isolation of the mini insertions IS 6 and IS 7 of E. coli

Abstract: The mini IS elements IS6 and IS7 have been detected in constitutive gal + revertants of galOP-308::IS2 (I), in which the expression of the gal operon is turned off by IS2 in orientation I. Both, IS6 and IS7, are integrated into IS2 proximal to the gal structural genes. IS6 is 115 base pairs long and causes 50% constitutive expression of the gal genes. IS7 is only 65 base pairs long and the gal operon is expressed 20% constitutively compared to the gal + wild type operon. Both IS6 and IS7 are excised frequently, in the absence of selective pressure. These findings are discussed with respect to the evolution of gene expression.

In vivo synthesis of rapidly-labelled RNA within the rat nodose ganglia following vagotomy.

Abstract: — The synthesis of rapidly-labelled RNA was studied in the nodose ganglia following unilateral vagotomy Early changes were detected in the electrophoretic patterns of RNA from both ganglia following a crush of the right vagus The right ganglionic response is characterized by two phases, the first involving rapid processing of rRNA with associated changes in ‘message-like’ RNA (mlRNA) A second change, detected by 14 days, appears to involve an increase in the heterodispersity of mlRNA and decreased processing of rRNA The left ganglion follows a very similar response to that of the right with the exception of the changes in rRNA However, the left response lags behind that of the right by at least 1 day We conclude that extensive changes in gene expression are occurring in both ganglia The similarity of the responses of both ganglia suggests that axon regeneration (in the right) and collateral sprouting (in the left) may produce analogous changes in gene expression, but not in rRNA synthesis