mRNA Start Point Research Articles

An improved primer extension method for detection of mRNA start-points using non-radioactive digoxigenin-labeling primers

An improved primer extension method using non-radioactive digoxigenin (DIG)-labeling primers is described which uses a commercially-available DIG-labeling and detection system to perform alternative hybridization-based "labeling" procedure of DNA markers and DIG chemiluminescent detection assay. The time-consuming annealing step and relatively low specificity of conventional protocol are also improved considerably by an application of one-step primer/mRNA annealing procedure and subsequent high-temperature reverse transcription reaction. This new protocol is convenient, simple, cost-effective and safe, and can allow the detection of even low abundance mRNA start-points in enriched poly(A)(+) RNA samples.

Cloning, expression, pharmacology and tissue distribution of the mouse somatostatin receptor subtype 5.

The gene encoding the mouse somatostatin receptor subtype 5 has been isolated from a genomic library and the mRNA start point mapped to position -95 relative to the translational start codon. The promoter region is devoid of TATA and CAAT boxes but contains putative binding sites for AP-1, AP-2 and SP1 and response elements for glucocorticoids (GRE) and phorbol esters (TRE). The encoded receptor protein with a predicted molecular weight of 42.5 kDa is comprised of 385 amino acids and thus contains 22 and 21 amino acids more than rat and human counterparts. The extra amino acids are caused by another translational initiation codon located further upstream. In the region of overlap the mouse somatostatin receptor subtype 5 displays 96.7% sequence identity to the rat and 81.7% to the human homologue. Application of somatostatin-14 and -28 to human embryonic kidney cells expressing the recombinant receptor resulted in the inhibition of forskolin-stimulated adenylyl cyclase with comparable EC50 values. Consistent with the observed sequence relationship, the mouse somatostatin receptor subtype 5 displays a pharmacological profile that resembles the rat homologue more closely than the human counterpart. mRNA for the mouse somatostatin type 5 receptor has been detected in pituitary, kidney, spleen and ovary and, to a lesser extent, in brain, stomach, intestine and thymus but was not observed in heart, pancreas and liver.

Cloning and Characterization of the Promoter Region of the Human Keratinocyte Growth Factor Gene

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family of related proteins, is expressed by stromal fibroblasts and acts on epithelial cells in a paracrine fashion. To understand the mechanisms responsible for regulating normal KGF expression and how these might be altered in disease, the 5'-flanking region of this gene was cloned. The presence of two KGF transcription initiation sites was suggested by ribonuclease protection assay and confirmed by primer extension analysis. Examination of the genomic DNA sequence revealed the presence of the putative promoter sequences TATTTA and CCAAT, located 31 and 50 base pairs upstream, respectively, from the first of the two mRNA start points, and putative initiator sequences surrounding each transcription start site. Transient transfection into murine NIH/3T3 fibroblasts demonstrated that the region required for basal level KGF promoter activity was located between bases -225 and +190. Inclusion of sequences between -1503 and -775 markedly reduced promoter activation, indicating the presence of negative regulatory element(s) in this region. A similar pattern of promoter activation was detected in human fibroblasts and in murine C2C12 myoblasts. In contrast, no chloramphenicol acetyltransferase activity was observed in macrophages and epithelial and lymphoid cells transfected with the same constructs. Northern blot analysis revealed a strong correlation between KGF RNA expression and promoter activation in all cells tested. Activation of the KGF promoter could be induced by the proinflammatory cytokines interleukin 1 and interleukin 6 and by the adenylate cyclase activator forskolin. Taken together, these results indicate the existence of cis-acting element(s) responsible for selective activation of the KGF promoter only in cells that express KGF mRNA and may provide a mechanistic basis for KGF gene expression during inflammation.

The Salmonella typhimurium katF (rpoS) gene: cloning, nucleotide sequence, and regulation of spvR and spvABCD virulence plasmid genes.

The spv region of Salmonella virulence plasmids is essential for the development of a systemic infection in mice. Transcriptional activation of the spvABCD operon occurs during stationary growth phase and is mediated by the regulatory gene product SpvR. We have previously shown that expression of a spvRAB'-cat fusion in Escherichia coli was dependent on the katF (rpoS) locus which encodes an alternative sigma factor (sigma S). The katF gene from Salmonella typhimurium has been cloned, sequenced, and used to construct Salmonella katF mutants by allelic replacement. Using these mutants, we demonstrated by mRNA and gene fusion analyses that sigma S, in conjunction with SpvR, controls the transcription of the regulatory gene spvR. In a second series of experiments, we sought to clarify the relationship between sigma S and SpvR in the control of spvABCD transcription. It was shown that expression of a transcriptional spvAB'-lacZ fusion could be restored in E. coli and Salmonella katF mutants when spvR was expressed in trans from an exogenous promoter. Moreover, identical spvA mRNA startpoints were detected in katF+ and katF strains. These results indicate that the reduction of spvABCD transcription in katF mutants is mainly due to decreased expression of spvR. Finally, mouse inoculation studies with S. typhimurium katF mutants of both wild-type and virulence plasmid-cured strains suggest that katF contributes to Salmonella virulence via the regulation of chromosomal genes in addition to that of spv genes.

Cloning and characterization of the gene encoding bovine mitochondrial adenylate kinase isozyme 3

The structure of the gene (AK3) encoding bovine mitochondrial (mt) adenylate kinase isozyme 3 (AK3) has been determined. The gene spans about 46 kb and splits into five exons. The 5'-flanking region of the gene lacks typical transcriptional regulatory elements such as TATA and CAAT boxes. The G + C content of this region is high (71%), and seven GC boxes are recognized. Possible sequences responsive to transcription factors, AP-1, AP-2, Myf-6 and MyoD1, are present in this region. In the 5'-flanking region, there are sequences (mt sequence) which were found in the genes encoding other mt proteins. Transcriptional mapping analyses revealed one major mRNA start point. The promoter activity measurement in Chinese hamster ovary cells by transient expression of the cat gene connected with the 5'-flanking region, showed presence of a functional promoter.

Division genes in Escherichia coli are expressed coordinately to cell septum requirements by gearbox promoters.

The cell division ftsQAZ cluster and the ftsZ-dependent bolA morphogene of Escherichia coli are found to be driven by gearboxes, a distinct class of promoters characterized by showing an activity that is inversely dependent on growth rate. These promoters contain specific sequences upstream from the mRNA start point, and their -10 region is essential for the inverse growth rate dependence. Gearbox promoters are essential for driving ftsQAZ and bolA gene expression so that the encoded products are synthesized at constant amounts per cell independently of cell size. This mode of regulation would be expected for the expression of proteins that either play a regulatory role in cell division or form a stoichiometric component of the septum, a structure that, independently of cell size and growth rate, is produced once per cell cycle.

Efficient expression of the Saccharomyces cerevisiae glycolytic gene ADH1 is dependent upon a cis -acting regulatory element ( UASRPG) found initially in genes encoding ribosomal proteins

The glycolytic form of alcohol dehydrogenase (ADHI) is encoded by the ADHI gene of Saccharomyces cerevisiae. We found that efficient expression of the ADH1 gene requires a sequence between bp −635 and −615 with respect to the + mRNA start point; removal of this sequence reduced ADH1 mRNA levels 25-fold but did not affect carbon-source regulation. DNAaseI footprinting analysis of the ADH1 promoter revealed the specific protection of a perfect match to UAS RPG at −630 to −615. UAS RPG is thought to be responsible for activation of transcription, via binding of the translation upstream factor (TUF), of genes encoding components of the translational appaatus. In band retardation assays, the promoters for the elongation factor 1α-encoding genes ( TEF1 and TEF2) competed for binding of the protein to the copy of UAS RPG in the ADH1 promoter. We conclude that TUF is probably involved in activation of the bulk of ADH1 transcription. Further, we propose that TUF has a role in the activation of many or most glycolytic genes. If so, it is essential for efficient expression of a wide variety of functionally disparate products that are required by yeast cells for rapid growth.

Mapping of the puh Messenger RNAs from Rhodospirillum rubrum: Evidence for tandem promoters

Abstract The mRNA transcripts of Rhodospirillum rubrum gene puh, coding for the H subunit of the photoreaction center, and of genes flanking puh were analyzed by blot hybridization. Open reading frame G115, upstream of structural gene puh, is transcribed as a 2.25-kilobase mRNA. Gene puh itself is transcribed as two mRNAs of 1118 and 1032 nucleotides. Mung bean nuclease protection analysis shows that the puh transcripts have different 5' termini within open reading frame G115 and a unique rho-independent termination signal within open reading frame I2372. The lifetimes of the puh messages, as determined by an oxygen blockade of transcription, were 10 and 12 min for the large and small puh mRNAs, respectively. An expression vector carrying a chloramphenicol acetyltransferase gene was used to select promoters in DNA stretches upstream of the startpoints of each of these transcripts. Chloramphenicol resistance was expressed in Escherichia coli, using as a promoter a 179-nucleotide stretch upstream of the small mRNA startpoint but not from a 124-nucleotide stretch upstream of the large mRNA startpoint. The promoter for the small mRNA, designated Ppuh2, is thought to encompass in its -10 and -35 regions a sigma 70-like RNA polymerase recognition sequence. The region upstream of the large message startpoint contains a sequence similar in its -12 and -24 regions to promoter sequences recognized by the sigma 60 RNA polymerase holoenzyme. This is designated as promoter Ppuh1.Ppuh1 is proposed to be strictly regulated by light intensity and by oxygen tension while Ppuh2 would be less sensitive to these parameters.

IRA1, an Inhibitory Regulator of the RAS-Cyclic AMP Pathway in Saccharomyces cerevisiae

A mutation in the gene IRA1 (formerly called PPD1) was originally characterized as a deficiency of a phosphoprotein phosphatase. The IRA1 gene has been cloned and sequenced. A large open reading frame (8,817 base pairs) which can encode a protein of 2,938 amino acids was found. Northern (RNA) blot analysis detected a message of about 10 kilobases, and nuclease S1 protection demonstrated mRNA start points at 97 and 98 base pairs upstream from the putative initiator ATG codon. Disruption of the IRA1 gene resulted in sensitivity to nitrogen starvation and heat shock. Diploids homozygous for the disrupted IRA1 gene were deficient in sporulation. Disruption of the IRA1 gene suppressed the lethality of the cdc25 mutation but did not suppress the lethality of either the ras1 ras2 or the cyr1 mutations. Deficiency of the phosphoprotein phosphatase was not reproducible in the disruption mutant of the IRA1 gene. Moreover, the ira1 mutant showed an increased level of cyclic AMP. Our results suggest that the IRA1 protein inhibits the function of the RAS proteins in a fashion antagonistic to the function of the CDC25 protein in the RAS-cyclic AMP pathway in Saccharomyces cerevisiae.

Nucleotide sequence analysis and expression studies of a chloramphenicol-acetyltransferase-coding gene from Clostridium perfringens

The nucleotide sequence of a Cm R determinant, lo cated on the Clostridium perfringens plasmid pIP401, was determined and its gene product was identified as chloramphenicol acetyltransferase (CAT). The cat structural gene is preceded by transcription-initiation signals characteristic for Escherichia coli σ 70 or Bacillus subtilis σ 43 promoters. By promoter probing in the heterologous hosts the direction of transcription of the clostridial cat gene was analysed and the cat mRNA start point was determined in vitro using the RNA polymerases of E. coli and B. subtilis. Comparison of the amino acid sequences of C. perfringens CAT and other CAT proteins of Gram-positive and Gram-negative origin shows a remarkable degree of homology between the various enzymes.

IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae.

A mutation in the gene IRA1 (formerly called PPD1) was originally characterized as a deficiency of a phosphoprotein phosphatase. The IRA1 gene has been cloned and sequenced. A large open reading frame (8,817 base pairs) which can encode a protein of 2,938 amino acids was found. Northern (RNA) blot analysis detected a message of about 10 kilobases, and nuclease S1 protection demonstrated mRNA start points at 97 and 98 base pairs upstream from the putative initiator ATG codon. Disruption of the IRA1 gene resulted in sensitivity to nitrogen starvation and heat shock. Diploids homozygous for the disrupted IRA1 gene were deficient in sporulation. Disruption of the IRA1 gene suppressed the lethality of the cdc25 mutation but did not suppress the lethality of either the ras1 ras2 or the cyr1 mutations. Deficiency of the phosphoprotein phosphatase was not reproducible in the disruption mutant of the IRA1 gene. Moreover, the ira1 mutant showed an increased level of cyclic AMP. Our results suggest that the IRA1 protein inhibits the function of the RAS proteins in a fashion antagonistic to the function of the CDC25 protein in the RAS-cyclic AMP pathway in Saccharomyces cerevisiae.

Cloning and analysis of a yeast genomic DNA sequence capable of directing gene transcription in Escherichia coli as well as in yeast.

A DNA fragment was isolated from yeast genomic sequences by its ability to direct the transcription of promoterless CmR (cat) gene in Escherichia coli and in yeast. Nucleotide sequencing and primer extension analysis showed that yeast DNA contains sets of consensus sequences pertaining to prokaryotic and yeast-type promoter elements. It was designated as yeast- and E. coli-type promoter (YEP1). Typical E. coli-type promoter elements are found at appropriate positions: TATTTT from -12 to -7 and TTGTCC from -35 to -30 with their spacing of 17 bp from the single mRNA start point determined by the primer extension. Analysis of cat transcripts from yeast cells showed that the YEP1 caused multiple transcription initiations at more than 20 different points that are spaced over a 100-bp region. The DNA is composed of A + T-rich sequences and putative TATA-like sequences are found at several places upstream from the transcription start points. Deletion analysis showed that a 276-bp sequence between -872 and -596 from the initiating ATG codon was required for the maximal promoter activity in yeast but not in E. coli.

Nucleotide sequence of the CLS4 (CDC24) gene of Saccharomyces cerevisiae

The nucleotide sequence of the CLS4 gene controlling Ca 2+ regulatory process of bud emergence, which was cloned previously [Ohya et al., J. Bacteriol. 165 (1986) 28–33], was determined. The CLS4 (CDC24) locus encodes a protein consisting of 736 amino acid (aa) residues with an M r of 83 970. By primer extension mapping, the mRNA start point was located 139 bp upstream from the translation start codon. The predicted CLS4 protein was hydrophilic with two serine + threonine-rich domains in the middle and C-terminal regions. It has two putative Ca 2+-binding regions, one being partly homologous to the Ca 2+-binding domain of the S-100a protein and the other that of α-lactalbumin.

Structure of the Bradyrhizobium japonicum gene hemA encoding 5-aminolevulinic acid synthase

The nucleotide (nt) sequence of the hemA gene, which encodes 5-aminolevulinic acid synthase (ALAS) from the bacterium Bradyrhizobium japonicum, is presented. This sequence predicts a protein of 408 amino acids (aa) with an M r of 44 599. This predicted amino acid sequence is highly homologous to that of the chicken embryonic liver ALAS, exhibiting a 48.8% identical amino acid sequence over the entire length of the bacterial protein. A single mRNA start point was demonstrated by S1 protection analysis for the B. japonicum hemA. The 5' end of the transcript is 100 nt upstream from the start codon. The sequence of the promoter region has some sequence homology to the −35 nt region of the Escherichia coli consensus promoter sequence but not to the −10 nt region. There is also one block of 9 nt found in both the B. japonicum glnA and hemA promoter regions.

Construction of LYS2 cartridges for use in genetic manipulations of Saccharomyces cerevisiae

Linker arrays were added to the 5' and 3' boundaries of the Saccharomyces cerevisiae LYS2 gene, which allow the generation of 18 LYS2 cartridges with different sticky ends. As it was necessary to define the beginning and the end of the approx. 4.5-kb LYS2 gene, we sequenced 1 kb of its 5' and 1.5 kb of its 3' region and mapped the mRNA start point. The open reading frame (ORF) found by this analysis was proven to be the LYS2 ORF by exchanging the sequences upstream from the presumptive ATG with the S. cerevisiae CYC1 promoter and subsequent demonstration of LYS2 expression in vivo. The proper functioning of the LYS2 cartridges was demonstrated by the transformation of lys2 mutant strains to Lys + prototrophy using plasmids furnished with a LYS2 cartridge.

Streptomyces contain Escherichia coli-type A + T-rich promoters having novel structural features

We describe here the isolation and characterization of a class of A + T-rich transcriptionally active sequences in the filamentous antibiotic-producing Gram-positive bacterial genus Streptomyces. These regions, which digress dramatically in base composition from the 73% G + C composition characteristic of the Streptomyces genome, promote gene expression in both Escherichia coli and Streptomyces lividans and contain the major elements that determine promoter strength in E. coli. The Streptomyces-E. coli-type promoters (SEP) also show novel structural features that include multiple direct repeats within the promoter region as well as a specific hexameric sequence in the vicinity of the mRNA start point.

Mannitol-specific enzyme II of the bacterial phosphotransferase system. III. The nucleotide sequence of the permease gene.

The nucleotide sequence of the mtlA gene, which codes for the mannitol-specific Enzyme II of the Escherichia coli phosphotransferase system, is presented. From the gene sequence, the primary translation product is predicted to consist of 637 amino acids (Mr = 67,893). This result is compared to the amino acid composition and molecular weight of the purified mannitol Enzyme II protein. The hydrophobic and hydrophilic properties of the enzyme were evaluated along its amino acid sequence using a computer program (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). The computer analysis predicts that the NH2-terminal half of the enzyme resides within the membrane, whereas the COOH-terminal half of the enzyme has the properties of a soluble protein. The possible functions of such a protein structure are discussed. RNA mapping has identified the promoter and mRNA start point for the mtl operon.