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PubMed1998

Cloning of two novel growth hormone transcripts expressed in human placenta.

Several isoforms of human GH (hGH) are produced by two related genes expressed in the pituitary (hGH-N) and in the placenta (hGH-V). These genes consist of five exons (denoted 1-5) separated by four introns (denoted A-D). In the present report, two new transcripts of the hGH-V gene are described. The coding region of the hGH-V gene was amplified by RT-PCR using placental complementary DNA as template. DNA sequencing of several clones revealed two novel transcripts. One had a 45-bp deletion caused by the use of an alternative splice acceptor site within exon 3, similar to that in the hGH-N gene, predicting a 20-kDa isoform of hGH-V. The other transcript was generated by the use of an alternative splice donor site causing a 4-bp deletion in the end of exon 4, predicting a 24-kDa protein with 219 amino acids, which we refer to as hGH-V3. The carboxy-terminal sequence of hGH-V3 differs from 22-kDa hGH-V and hGH-V2, the two previously reported transcripts of the hGH-V gene, and does not contain a predicted transmembrane domain as described for hGH-V2. Ligase chain reaction was then used to analyze the possible use of the same splicing pattern in transcripts derived from the other genes of the hGH-gene cluster. Alternatively spliced transcripts encoding the 20-kDa hGH isoform were detected from the hGH-N and hGH-V genes, but not from the human chorionic somatomammotropin-A/B genes. The alternative splicing generating hGH-V3 was only demonstrated in transcripts derived from the hGH-V gene. Using competitive RT-PCR, the expression of hGH-V3 was estimated to be 10% of the hGH-V messenger RNA in full-term normal placentas and in placentas from pathological pregnancies. The 20-kDa hGH-V was detected in two of four full-term normal placentas, whereas a weak signal was observed in one of the pathological placentas. We conclude that the hGH-V primary transcript undergoes alternative splicing pathways generating at least four different messenger RNAs, predicting the expression of different hGH isoforms, including two with a complete sequence divergence in the carboxy-terminus.

Alternative Splicing↗
PubMed1990

Characterization of transcriptional initiation from promoters P1 and P2 of the pyrBI operon of Escherichia coli K12.

Expression of the pyrBI operon of Escherichia coli K12, which encodes the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is negatively regulated by pyrimidine availability, primarily through an attenuation control mechanism, and also appears to be subject to stringent control. Previous in vitro transcription studies indicated that the pyrBI operon is transcribed from tandem promoters designated P1 and P2, which appeared to be of similar strength. In this study, we characterized these promoters in detail and examined their role in pyrBI expression. Our results show that although transcription is initiated at both promoters in vivo, greater than 99% of the pyrBI transcripts are initiated at promoter P2, indicating that this is the only physiologically significant promoter. The level of transcripts initiated at promoter P2 was found to be higher in cells grown under pyrimidine-limiting conditions compared to that in cells grown under conditions of pyrimidine excess, indicating pyrimidine-mediated regulation at the level of transcriptional initiation. In vitro characterization of transcription from the pyrBI promoter region showed that non-physiological reaction conditions used in the original identification of the two promoters greatly overestimated the strength of promoter P1. Further in vitro characterization of the two promoters showed that transcription from promoter P2, but not from promoter P1, is inhibited by guanosine tetraphosphate and exhibits salt and heparin sensitivity typical of a stringently controlled promoter. In addition, heparin-challenge experiments revealed a UTP-induced instability of transcriptional initiation complexes at promoter P2 which may be of regulatory significance.

Amino Acid Sequence↗
PubMed1991

Retrotransposons and evolution in phlebotomines.

The polymerase chain reaction was used to amplify a segment of the reverse transcriptase (RT) gene of putative retrotransposons from Phlebotomus (Larroussius) perniciosus, P. (L.) perfiliewi, P. (Phlebotomus) papatasi and Lutzomyia (Lutzomyia) longipalpis. Based on amino acid sequence comparisons with known RT genes, the amplified products of these species were shown to be derived from non-LTR retrotransposons related to the F element of Drosophila melanogaster. The usefulness of this technique is discussed in relation to taxonomy and genetic manipulation.

Amino Acid Sequence↗
PubMed1995

The differential processing of homodimers of reverse transcriptases from human immunodeficiency viruses type 1 and 2 is a consequence of the distinct specificities of the viral proteases.

Active, recombinant p68 reverse transcriptase (RT) from human immunodeficiency virus type 2 (HIV-2), with an NH2-terminal extension containing a hexahistidine sequence was isolated from extracts of Escherichia coli by immobilized metal affinity chromatography. Treatment of the purified p68/p68 homodimer of HIV-2 RT with recombinant HIV-2 protease generates stable, active heterodimer (p68/p58) that is resistant to further hydrolysis. Analysis of this p68/p58 HIV-2 RT heterodimer revealed that while one subunit is intact p68, the p58 subunit is COOH-terminally truncated by cleavage, not at Phe440 as is seen in processing of the p66/p66 HIV-1 RT homodimer by HIV-1 protease, but at Met484. The expected COOH-terminal p10 fragment resulting from hydrolysis of p68 at Met484 is not released intact, but undergoes further cleavage at Asn494, Met503, and Tyr532. Processing of p68/p68 HIV-2 RT with the HIV-1 protease led to cleavage of the Phe440-Tyr441 bond, exactly as is seen with p66/p66 HIV-1 RT, to give the analogous p53 subunit. Studies of a peptide substrate modeled after residues 437-444 in HIV-2 RT showed that while the HIV-1 protease was able to cleave the Phe440 bond, this bond was resistant to cleavage by the HIV-2 enzyme. Our findings provide a rationale for the previous observation that the RT heterodimer isolated from HIV-2 lysates is larger than that from HIV-1. We conclude that the p68/p58 HIV-2 RT heterodimer, containing the Met484 truncated p58 subunit, is a biologically relevant form of the enzyme in vivo.

Amino Acid Sequence↗
PubMedDate not supplied

Coding region of segment A sequence of a very virulent isolate of IBDV--comparison with isolates from different countries and virulence.

We determined the sequence of the coding region of segment A, coding for the viral proteins (VPs) VP2, VP4, and VP3, of a very virulent (vv) infectious bursal disease virus (IBDV) isolated in Israel and named IBDVks. We compared the deduced amino acid sequences of the proteins of the new isolate with those of the same proteins from several IBDV isolates, as published in recent years. The amino acid sequences of VP3 and VP4 of the Israeli isolate were 1.9%-2.3% different from the sequences of their counterparts from classical strains. Thus, the stable region of VP2 of IBDVks was very similar (0-0.68% difference) to the same region of VP2 from vv strains from Europe and Japan but distinct from that of proteins from classical strains from Europe, the United States, and Australia (up to 9.42% divergence), showing that IBDVks is more closely related to the vv strains from Europe and Japan. We found that viruses isolated in recent years resemble each other more than isolates from the same areas isolated a few years earlier. Hence, IBDVks can be categorized in one group with vv new isolates from Europe and Japan. This group has been found to be distinct from new isolates in the United States and strains isolated before the IBDV epidemic during the late 1980s.

Animals↗
PubMed1998

Antibiotic resistance in mycobacteria.

Multiple drug resistance in mycobacteria compromises the use of antibiotics. Although the genetic and biochemical bases of antibiotic resistance in mycobacteria are largely understood, a number of questions remain to be addressed. This chapter discusses the potential roles of hypermutability and compensatory mutations in establishing stable resistant phenotypes in the pathogenic mycobacteria.

Amino Acid Sequence↗
PubMed1991

The muscle specific domain of mouse N-CAM: structure and alternative splicing patterns.

The neural cell adhesion molecule (N-CAM) is an important mediator of calcium independent cell-cell interactions. Variations in the primary structure of the protein are due to alternative splicing of pre-mRNA in the region encoding the extracellular, trans-membrane and cytoplasmic domains. In order to identify the patterns of exon usage during development of skeletal muscle and brain of the mouse, a coupled reverse-transcriptase/polymerase chain reaction was used to identify the murine homologues of the muscle-specific domain (MSD), located between exons 12 and 13 in human N-CAM mRNA. The cDNAs produced have been cloned and sequenced, or analysed directly. The amplification reactions were shown to maintain the concentration ratios of the initial cDNAs. The results indicate that the mouse homologue to exon MSD1a is under tissue and developmental regulation that is independent of exons MSD1b and MSD1c. The inclusion of the triplet exon AAG is also regulated in a cell- and stage-specific manner, which is independent of the other alternatively spliced exons of this domain.

Animals↗
PubMed1986

Promoter used by sigma-29 RNA polymerase from Bacillus subtilis.

Gene expression during endospore formation by Bacillus subtilis is controlled in part by a sporulation-induced form of RNA polymerase, E sigma 29. The determination of the nucleotide sequences that govern utilization of promoters by E sigma 29 has been limited by the small number of available promoters that are recognized by E sigma 29. In the present report we describe a promoter that is adjacent to the rrnB region of the B. subtilis chromosome and is utilized in vitro and in vivo by E sigma 29. S1 nuclease mapping and dinucleotide priming experiments have been used to determine the start point of transcription. The nucleotide sequences near the -10 and -35 region of this promoter, bvx, are conserved, and resemble sequences at these regions for other promoters that are utilized by E sigma 29.

Bacillus subtilis↗
PubMedDate not supplied

[Structure-activity organization of the variola virus genome. III. Sequencing and analysis of the nucleotide sequence of the conserved region of HindIII-F, -N-, and -A-fragments of the India 1967 strain genome].

Computer analysis of variola major virus (VAR) genomic fragment bounded by open reading frames (ORFs) D1R and A33L which is 47,961 bp long revealed 46 potential ORFs. The VAR proteins were compared with the analogous proteins of vaccinia virus strain Copenhagen. The subunits of DNA-dependent RNA polymerase, as well as the transcription factors, mRNA capping enzymes, and proteins necessary for the virion morphogenesis proved to be highly conservative within orthopoxviruses. The most pronounced differences between the VAR genome fragment under study and the corresponding vaccinia virus fragment were revealed in the vicinity of the gene encoding the A-type inclusion body protein. The possible functions of the analyzed viral proteins are discussed.

Amino Acid Sequence↗
PubMed1995

Genetic and transcriptional organization of the region encoding the beta subunit of Bacillus subtilis RNA polymerase.

The gene encoding the beta subunit of Bacillus subtilis RNA polymerase was isolated from a lambda gt11 expression library using an antibody probe. Gene identity was confirmed by the similarity of its predicted product to the Escherichia coli beta subunit and by mapping an alteration conferring rifampicin resistance within the conserved rif coding region. Including the rif region, four colinear blocks of sequence similarity were shared between the B. subtilis and E. coli beta subunits. In E. coli, these conserved blocks are separated by three regions that either were not conserved or were entirely absent from the B. subtilis protein. The B. subtilis beta gene was part of a cluster with the order rplL (encoding ribosomal protein L7/L12), orf23 (encoding a 22,513-dalton protein that is apparently essential for growth), rpoB (beta), and rpoC (beta'). This organization differs from the corresponding region in E. coli by the inclusion of orf23. Experiments using promoter probe vectors and site-directed mutagenesis located a major rpoB promoter overlapping the 3'-coding region of orf23, 250 nucleotides upstream from the beta initiation codon. Thus, the B. subtilis rpoB region differs from its E. coli counterpart in both genetic and transcriptional organization.

Amino Acid Sequence↗
PubMed1996

The Mfd protein of Bacillus subtilis 168 is involved in both transcription-coupled DNA repair and DNA recombination.

Inactivation of Bacillus subtilis orf1177 in an otherwise Rec+ strain reduced genetic exchange and DNA repair. When the mutation was transferred into a set of recombination-deficient and repair-deficient strains, the DNA repair and recombination ability of the double or triple mutant strains was drastically reduced. B. subtilis Orf1177 protein shares substantial homology with the Escherichia coli Mdf, RecG and UvrB proteins. In vivo analysis of UV-induced mutations suggests that Orf1177 is necessary for strand-specific DNA repair, as is the case for the E. coli MFD protein. Therefore, orf1177 and Orf1177 were termed mfd gene and Mfd protein, respectively. The purified Mfd protein has a native molecular mass of 140 kDa (expected molecular mass 133 kDa). The Mfd protein is a sequence-independent DNA binding protein with weak ATPase activity. The Mfd protein was able to displace in vitro B. subtilis or E. coli RNA polymerase stalled at a lesion. Therefore, Mfd protein appears to target the transcribed strand for repair by recognizing a stalled RNA polymerase and dissociating it from the DNA. In addition, the strong recombination-deficient phenotype of mfd- rec- strains suggest that Mfd protein is involved in homologous DNA recombination.

Amino Acid Sequence↗
PubMed1997

FHIT gene and the FRA3B region are not involved in the genetics of renal cell carcinomas.

The FHIT gene locus at 3p14.2 covers about 500 kb, including the fragile site FRA3B and the constitutional t(3;8) breakpoint associated with the development of multiple renal cell carcinomas (RCC). A terminal deletion of the short arm of chromosome 3 with the most distal breakpoint in the FRA3B region is the characteristic genetic event in nonpapillary RCC. Since aberrant FHIT transcripts have been observed in gastrointestinal and other tumors, this gene has been suggested to function as a tumor suppressor. To evaluate the role of FHIT and the FRA3B region in the genetics of RCC, we analyzed FHIT expression by RT-PCR and performed microsatellite deletion mapping in the FHIT region. In addition to two cases from a t(3;8) family, only three out of 100 sporadic nonpapillary RCC showed a breakpoint within the FHIT region, whereas 94 tumors showed a deletion breakpoint proximal to the FHIT gene. FHIT transcripts of normal size were observed in 33 out of 34 tumors. Direct sequencing of eight PCR products revealed a normal FHIT sequence without mutations in the coding region. An established cell line from a renal cancer xenograft showed a smaller FHIT transcript. Sequence analysis revealed a mixture of several splicing variants of the FHIT gene. Since only three out of 100 sporadic nonpapillary RCC had a deletion breakpoint within the FRA3B/FHIT region, and since all but one renal cell tumor showed a normal FHIT transcript, we can exclude the involvement of the FHIT gene and the FRA3B region in the genetics of renal cell cancer.

Acid Anhydride Hydrolases↗
PubMed1990

Identification of the vaccinia virus gene encoding an 18-kilodalton subunit of RNA polymerase and demonstration of a 5' poly(A) leader on its early transcript.

The DNA-dependent RNA polymerase of vaccinia virus contains 8 to 10 virus-encoded polypeptides. We have mapped the gene encoding an 18-kilodalton RNA polymerase subunit to D7R, the seventh open reading frame of the HindIII D genomic subfragment. Localization of this gene was achieved by using antibody to the purified RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments. The identification was confirmed by translation of D7R transcripts made in vitro with bacteriophage T7 RNA polymerase. The phenotypes of two previously isolated conditionally lethal temperature-sensitive mutants that map to D7R (J. Seto, L. M. Celenza, R. C. Condit, and E. G. Niles, Virology 160:110-119, 1987) are consistent with an essential role of this subunit in late transcription. This polymerase gene, designated rpo18, predicts a polypeptide of 161 amino acids with a molecular mass of 17,892. The rpo18 gene is transcribed early in infection, even though the 5'-TAAATG-3' motif, which is conserved among many genes of the late class, is present near the RNA start site. Characterization of the 5' end of the early transcript by several different methods, including cDNA cloning, revealed a poly(A) leader with up to 14 adenylate residues, whereas only 3 are present in the corresponding location of the DNA template. Similar but somewhat longer poly(A) leaders have previously been observed in mRNAs of late genes. We noted a TAAATG motif near the initiation site of several other early genes, including the viral DNA polymerase, and carried out additional experiments to demonstrate that their early transcripts also have 5' poly(A) leaders. Thus, formation of the poly(A) leader is not exclusively a late function but apparently depends on sequences around the transcription initiation site.

Base Sequence↗
PubMed1997

30 years later--a new approach to Sol Spiegelman's and Leslie Orgel's in vitro evolutionary studies. Dedicated to Leslie Orgel on the occasion of his 70th birthday.

The conditions necessary for evolution are amplification, mutagenesis and selection. Here we describe the evolutionary response of an in vitro replicating system to the selection pressure for fast growth and show what happens to the amplified molecules within this replication system. Our emphasis is on methodology, on the monitoring and the automation of experiments in molecular evolution. In order to perform in vitro studies on the evolution of RNA molecules, a modified self-sustained sequence replication (3SR) method was used. In the first step of the 3SR reaction, the RNA template is reversely transcribed by HIV-1 reverse transcriptase, followed by a second strand synthesis and the transcription of the resulting dsDNA by T7 RNA polymerase. The selection pressure (fast growth) was achieved by applying the principle of serial transfer pioneered in the laboratories of Sol Spiegelman and Leslie Orgel. At the end of the exponential growth phase of the 3SR reaction, an aliquot of the reaction mixture is transferred into a new sample containing only buffer, nucleotides and enzymes while RNA template molecules are provided by the transfer. The conditions in the exponential growth phase allow the RNA molecules to be amplified in a constant environment; all enzymes (HIV-1 reverse transcriptase and T7 RNA polymerase) and nucleotides are present in large excess. Therefore, transferring reproducibly within the exponential growth phase is equivalent to selecting for fast growth; those molecules which can replicate faster will displace others after several transfers. The experiments were performed using a serial transfer apparatus (STA) which allows the nucleic acid concentration to be monitored on-line by measuring the laser-induced fluorescence caused by intercalation of thiazole orange monomers into the RNA/DNA amplification products. The serial transfer experiments were carried out with an RNA template (220b RNA) that represents a 220-base segment of the HIV-1 genome and comprises the in vivo primer binding site (PBS) for the HIV-1 reverse transcriptase. It could be shown that after only two serial transfers two RNA species (EP1 and EP2) emerged that were much shorter. EP1 (48b) and EP2 (54b) were formed by deletion mutations within the original 220b RNA template in the very beginning of the serial transfer experiment; due to their higher replication rate (calculated from the growth curves derived on-line) these two deletion mutants displaced the original 220b RNA template in the course of the following thirty transfers. We assume that these two RNA species evolved independently of each other. Their formation was probably induced by a strand-transfer reaction of HIV-1 reverse transcriptase. Sequence analyses of these two evolution products seem to confirm such a presented pathway. 30 years after Spiegelman's experiment, the study described here is another answer to the question he posed: 'How do molecules evolve if the only demand is the biblical injunction: multiply?'. The answer, derived from a modified 3SR amplification system (mimicking a part of the HIV-1 replication cycle in vitro), is the same as thirty years ago: The RNA molecules adapt to the new conditions by throwing away any ballast not needed for fast replication. Clearly, this is only one aspect of molecular evolution; however, it shows that we should be careful in designating unidentified genetic material as 'junk DNA'.

Bacteriophage T7↗
PubMed1998

A surface of Escherichia coli sigma 70 required for promoter function and antitermination by phage lambda Q protein.

The sigma initiation factor sigma70 of Escherichia coli acts not only in promoter recognition and DNA strand opening, but also to mediate the transformation of RNA polymerase (RNAP) to an antiterminating form by the phage lambda gene Q protein. Q is able to bind and modify RNAP when alpha70, still present in the initially elongating enzyme, recognizes a repeat of the -10 promoter element and induces a transcription pause. We have isolated mutations in the rpoD gene for sigma70 that impair Q function because they reduce the ability of sigma70 to recognize the downstream pause site. These mutations identify a locus of sigma70 that is important for the formation and stability of open promoter complex, likely because it mediates protein interactions with RNAP core.

Amino Acid Substitution↗
PubMed1988

Characterization and promoter selectivity of Lactobacillus acidophilus RNA polymerase.

DNA-dependent RNA polymerase has been purified from gram-positive Lactobacillus acidophilus and found to be composed of 4 protein subunits, alpha, beta, beta', and sigma, with molecular weights of 40,000, 150,000, 135,000, and 45,000 kD, respectively, estimated on the basis of SDS-polyacrylamide gel electrophoresis. The purified enzyme exhibits optimal activity in the presence of Mn2+, while Mg2+ shows only a slight effect. The L. acidophilus enzyme transcribes several Escherichia coli promoters examined so far, such as promoters of trp operon, lacUV5, and bla P3 from pBR322, whereas it lacks the ability to recognize bla P1 and tet P2 promoters from pBR322. Thus, the specificity of L. acidophilus RNA polymerase in recognizing the promoters is somehow different from that of the E. coli enzyme. By means of an in vitro transcription assay system for L. acidophilus RNA polymerase, 2 promoters have been identified in the DNA of an L. acidophilus cryptic plasmid (pRNL5). These promoters possess nucleotide sequences in the -10 region similar to the consensus sequence for the E. coli promoters.

Animals↗
PubMed1984

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. VI. A redundant region in the beta polypeptide.

A single species of RNA polymerase is responsible for the transcription of genetic material in Escherichia coli; and, each component of this essential enzyme is encoded by a single gene. Nevertheless, we report the characterisation of a viable deletion mutant lacking about 165 bp in the region coding for residues 965 to 1143 of the beta subunit of this enzyme. The foreshortened beta polypeptide appears to function as normal, suggesting that this region is, in fact, non-essential with regard to the usual functions of E. coli RNA polymerase.

Amino Acid Sequence↗
PubMed1987

Interactions of a proteolytically nicked RNA polymerase of bacteriophage T7 with its promoter.

The association of nicked RNA polymerase of bacteriophage T7 (Ikeda, R. A., and Richardson, C. C. (1987) J. Biol. Chem. 262, 3790-3799) with the T7 phi 10 promoter has been examined by DNA cleavage protection. The phi 10 promoter consists of a 23-base pair consensus sequence that extends from -17 to +6 with respect to the site of the initiation of transcription (+1). Nicked T7 RNA polymerase alone protects 20 bases from -21 to -2 (+/- 1) base at each border. Initiation and synthesis of the trinucleotide r(GGG) expands and shifts the sequence protected by nicked T7 RNA polymerase. Twenty-five bases are protected from -17 to +8 (+/- 1). The polymerization of three additional ribonucleotides, synthesis of the hexamer r(GGGAGA), further expands the protected sequence. Twenty-seven bases are protected from -17-+10 (+/- 1). Finally, the synthesis of a pentadecaribonucleotide transcript, r(GGGAGACCACGG), leads to the formation of a transcription complex that protects 22 bases from -2-+20 (+/- 1). In comparison to the sequences protected by T7 RNA polymerase the sequences protected by the nicked enzyme are shortened at the 5' end and are translocated downstream much earlier during the initiation of transcription. It appears that a portion of the DNA contacts made at the amino terminus of T7 RNA polymerase are disrupted in the small fragment of nicked T7 RNA polymerase. The changes that are observed in the sequences protected by nicked T7 RNA polymerase are reflected in the physical characteristics of the DNA X enzyme complexes. The number of ion pairs formed by the r(GGG)-initiated complex of the nicked enzyme is reduced, and the association constant for the formation of the r(GGG)-initiated complex is decreased as compared to the intact T7 RNA polymerase.

Base Sequence↗