Transcription Unit Of Gene Research Articles

Efficient tissue‐specific expression of bovine α‐lactalbumin in transgenic mice

Transgenic mice carrying a 3.2-kb genomic fragment comprising the bovine alpha-lactalbumin gene transcription unit with 750 bp and 336 bp of 5' and 3' flanking regions, respectively, were generated. Tissue-specific expression of the transgene was observed in five out of the six lines analyzed at levels close to the endogenous gene in two of them.

Evolutionary conservation of homeodomain-binding sites and other sequences upstream and within the major transcription unit of the Drosophila segmentation gene engrailed.

The engrailed (en) gene functions throughout Drosophila development and is expressed in a succession of intricate spatial patterns as development proceeds. Normal en function relies on an extremely large cis-acting regulatory region (70 kilobases). We are using evolutionary conservation to help identify en sequences important in regulating patterned expression. Sequence comparison of 2.6 kilobases upstream of the en coding region of D. melanogaster and D. virilis (estimated divergence time, 60 million years) showed that 30% of this DNA occurs in islands of near perfect sequence conservation. One of these conserved islands contains binding sites for homeodomain-containing proteins. It has been shown genetically that homeodomain-containing proteins regulate en expression. Our data suggested that this regulation may be direct. The remaining conserved islands may contain binding sites for other regulatory proteins.

Transcription of the celE gene in Thermomonospora fusca

The steady-state level of celE mRNA (coding for cellulase E5) in Thermomonospora fusca YX was measured by Northern (RNA blot) hybridization under conditions causing induction or repression of cellulase synthesis. A good correlation was found between the mRNA level and the level of cellulase E5, suggesting that the T. fusca celE gene is regulated at the level of mRNA and, most likely, at the level of transcription. The 5' and 3' ends of the celE gene transcription unit were determined by S1 mapping with single-stranded DNA probes. These results showed that there were three species of celE mRNA in T. fusca YX with closely spaced 5' ends and identical 3' ends. The size of each mRNA was about 1.5 kilobases, from both the Northern and S1 data. This size is only slightly longer than that required to code for the 45-kilodalton E5 protein. In Escherichia coli D318 (celE), the 5' ends of the celE mRNAs are identical to those in T. fusca, but the 3' ends are located ca. 300 base pairs upstream of the T. fusca 3' end. The region where the putative celE promoters were located had some interesting features, including a 60-base-pair A + T-rich sequence and sequences resembling sigma 60 promoters.

Nucleotide sequence of the human placental alkaline phosphatase gene. Evolution of the 5′ flanking region by deletion/substitution.

Three closely related alkaline phosphatase (ALP) genes reside on the long arm of chromosome 2 in man. One of these genes (the placental ALP-1) encodes the classic heat-stable placental alkaline phosphatase. Another gene (the placental ALP-2) is closely related to the placental ALP-1 and may encode the so-called placental ALP-like enzyme of the testis and thymus. The third member of this gene family (the intestinal ALP gene) encodes the intestinal alkaline phosphatase. The expression of the placental ALP-1 and intestinal ALP genes is highly tissue-specific in spite of nearly 90% sequence similarity within their exons. To help determine the basis for this tissue specificity, the nucleotide sequence of the placental ALP-1 gene and some of its 5' flanking region has been determined and analyzed by comparison with placental ALP-2 and intestinal ALP gene sequences. The placental ALP-1 gene transcription unit has 4087 bases between the major cap site and the most distal of several reported 3' ends. The protein coding region is divided by 10 short introns varying in size from 74 to 241 nucleotides. Three of these introns bisect regions of the gene that encode residues conserved between the active site of the Escherichia coli enzyme and the human placental ALP. This result suggests that the human alkaline phosphatase genes have evolved in an intron-independent fashion. A comparison of the placental ALP-1 5' flanking sequence (up to -540) with the analogous sequence of the intestinal ALP gene revealed several deletion/substitutions which could be important in determining the tissue-specific expression of these genes.

Stage-specific selection of alternative transcriptional initiation sites from the 5C actin gene of Drosophila melanogaster.

The transcription unit of the 5C actin gene exhibits a complex organization that is unique among the six actin genes of Drosophila melanogaster. Three different mRNA size classes showing distinct patterns of accumulation throughout development are detected on Northern blots. We have determined the structure of the various 5C actin transcripts by exon mapping using strand-specific RNA probes, primer extension analysis, and DNA sequences analysis of both cDNA and genomic clones. All the transcripts share a single protein-coding nucleotide sequence but are heterogeneous in the 5' and 3' untranslated regions. The 5' untranslated region of each transcript consists of either one of two small exons (exon 1 and exon 2) which are alternatively spliced to a single acceptor site 8 bp upstream from the translation initiation codon in exon 3. Results from primer extension analysis suggest that transcription can initiate from either exon 1 or exon 2, and also from a third site within exon 2. We detect an increase in the relative abundance of exon 1-containing transcripts at larval and pupal stages, as well as a change in the proportion of transcripts that initiate at either of the two exon 2 sites. Five polyadenylation sites have been found within three termination/processing regions that define the three size classes of polyadenylated transcripts. The results of our experiments indicate the existence in vivo of all possible combinations of 5' exon with 3' polyadenylation site. However, particular combinations of 5' initiation site and 3' polyadenylation site are preferred at certain developmental stages.

Streptococcus pyogenes type 12 M protein gene regulation by upstream sequences

A partial nucleotide sequence that included 1,693 base pairs of the M12 (emm12) gene of group A streptococci (strain CS24) and adjacent upstream DNA was determined. Type 12 M protein-specific mRNA of strain CS24 is transcribed from two promoters (P1 and P3) separated by 30 bases. The transcription start sites of the emm12 gene were located more than 400 bases downstream of a deletion that causes decreased M-protein gene transcription in strain CS64. Deletion analysis of M protein-expressing plasmids indicated that an upstream region greater than 1 kilobase is required for M-protein gene expression. The M-protein gene transcriptional unit appears to be monocistronic. Analysis of the emm12 DNA sequence revealed three major repeat regions. Two copies of each repeat, A and B, existed within the variable 5' end of the gene; repeat C demarcated the 5' end of the constant region shared by emm12 and emm6.

Ontogenic expression of a CyI actin fusion gene injected into sea urchin eggs.

The 5' terminus of the CyI actin gene transcription unit of Strongylocentrotus purpuratus was located by primer extension and other procedures, and the flanking upstream region was partially sequenced and mapped. A fusion gene was constructed containing about 2.5 kb of 5' flanking sequence, the transcribed leader sequence, and the first few codons of the CyI gene ligated to the bacterial gene coding for chloramphenicol acetyl transferase (CAT). This was micro-injected into the cytoplasm of S. purpuratus eggs, and CAT enzyme activity was measured at various stages of embryonic development. CAT synthesis was activated between 10 and 14 h postfertilization, the same time at which newly synthesized transcripts of the endogenous CyI gene first appear. The exogenous CyI.CAT fusion DNA replicated actively during cleavage, as observed previously for other DNAs injected into sea urchin egg cytoplasm. Thus the absence of CAT activity prior to 10 h postfertilization could not be due to insufficient CyI.CAT genes. The amounts of CAT enzyme produced by embryos bearing CyI.CAT deletions that lack various regions of the CyI sequence were measured. As little as 254 nucleotides of upstream CyI sequence suffice for correct temporal activation of the fusion construct, although the level of CAT enzyme produced in embryos bearing any deletion retaining less than 850 nucleotides of upstream sequence was significantly lowered compared to controls bearing the complete CyI.CAT fusion construct.

DNA inversion within the apolipoproteins AI/CIII/AIV-encoding gene cluster of certain patients with premature atherosclerosis.

The genes coding for apolipoproteins (apo) AI, CIII, and AIV, designated APOA1, APOC3, and APOA4, respectively, are closely linked and tandemly organized in the long arm of the human chromosome 11. A DNA rearrangement involving the genes encoding apoAI and apoCIII in certain patients with premature atherosclerosis has been associated with deficiency of both apoAI and apoCIII in the plasma of these patients. Structural characterization of the genes for apoAI and apoCIII in one of these patients indicates that this rearrangement consists of a DNA inversion containing portions of the 3' ends of the apoAI and apoCIII genes, including the DNA region between these genes. The breakpoints of this DNA inversion are located within the fourth exon of the apoAI gene and the first intron of the apoCIII gene. Thus, this DNA inversion results in reciprocal fusion of the apoAI and apoCIII gene transcriptional units. Expression of these gene fusions in cultured mammalian cells results in stable mRNA transcripts with sequences representing fusions of the apoAI and apoCIII mRNAs. These results indicate that absence of transcripts with correct apoAI and apoCIII mRNA sequences causes apoAI and apoCIII deficiency in the plasma of these patients and suggest that these apolipoproteins are involved in cholesterol homeostasis and protection against premature atherosclerosis.

Porcine relaxin. Gene structure and expression.

A porcine genomic cosmid library was constructed to study the structure and regulation of the porcine relaxin gene. Two overlapping cosmids containing relaxin-specific sequences were isolated, and a 9-kilobase BamHI fragment containing the porcine relaxin gene was sequenced. The sequence derived from these cosmids was used to characterize the relaxin gene transcription unit utilized in the pregnant ovary. Analysis of chromosomal and cosmid DNA has shown that porcine relaxin is encoded by a single copy gene, comprising two exons separated by a 5.5-kilobase intron. The gene contains elements representing two classes of repeated sequences homologous with the human Alu and Kpn families. The transcriptional initiation site of this gene was located by primed extension and direct sequencing of relaxin-specific cDNA. Furthermore, the promoter elements were mapped and sequenced, and transcriptional activity was demonstrated. The promoter contained sequences with homology to regions conserved within retroviral long terminal repeats and within the adenovirus E4 promoter.

Erg, a human ets-related gene on chromosome 21: alternative splicing, polyadenylation, and translation.

The avian acute leukemia virus E26 induces a mixed erythroid-myeloid leukemia in chickens and carries two distinct oncogenes, v-myb and v-ets. Recently, a novel gene named erg, closely related to the v-ets oncogene, was identified in human COLO 320 cells and the nucleotide sequence of its approximately 5.0-kilobase transcript, erg 1 was determined. In the present study, the nucleotide sequence of the alternatively spliced transcript, erg 2, was found to differ from erg 1 by a splicing event that causes a coding frameshift near the amino terminus, resulting in an additional 99-amino acid insertion at the amino-terminus. Expression of complementary DNAs for the two transcripts in vitro resulted in synthesis of polypeptides of approximately 41 and 52 kilodaltons, suggesting the use of alternative translation initiation codons in the case of erg proteins. The erg gene was localized by somatic cell genetic analysis to human chromosome 21. It is proposed that alternative sites of splicing and polyadenylation, together with alternative sites of translation initiation, allow the synthesis of two related polypeptides from a single erg gene transcriptional unit.

Organization and expression of the Plasmodium knowlesi circumsporozoite antigen gene

To investigate the mechanisms regulating the stage specific expression of the Plasmodium knowlesi circumsporozoite (CS) antigen gene, the sporozoite genomic DNA copy of the CS gene has been isolated and compared with the blood stage genomic DNA and the sporozoite cDNA copies. The genomic DNA sequences of the two developmental stages are identical across 4 kilobase pairs of the chromosome containing the entire CS gene transcriptional unit. From restriction enzyme mapping no DNA rearrangements over 15 kilobase pairs of the chromosome containing the CS gene appear to be involved in its stage-specific expression and its regulation appears to be at the level of transcription or RNA stability. S1 nuclease and primer extension transcript mapping studies suggest that the CS mRNA has multiple start sites, that the leader sequence is devoid of introns, and is approximately 270 bases long. Consensus eukaryotic TATA and CAAT box sequences and potential regulatory elements, including sequences highly homologous to the reiterated and core enhancer sequences of SV40 precede the gene.

Evidence that DNA involved in the expression of nodulation (nod) genes in Rhizobium binds to the product of the regulatory gene nodD.

In Rhizobium leguminosarum biovar viciae, the regulatory nodulation nodD gene has at least two functions. It constitutively represses its own transcription and in the presence of inducer flavonoid molecules, it activates the expression of two other nod gene transcriptional units, nodABCIJ and nodFE. Upstream of nodA and nodF is a conserved sequence, the nod box, which has been implicated in nodD-mediated transcriptional activation of these genes. DNA fragments spanning the nod boxes that precede nodA and nodF were end-labelled and were exposed to cell-free extracts obtained from strains of Rhizobium. Using the gel retardation technique, it was shown that a complex between protein and these DNA fragments was formed, but only if the extract contained a functional nodD gene. Evidence that the protein that binds to the regulatory sequences is the nodD gene product came from the observation that a complex was formed between the nod box preceding nodA and protein from a cell-free extract isolated from Escherichia coli containing the cloned nodD gene. Extracts from Rhizobium strains containing mutant forms of nodD which were specifically affected in autoregulation or in flavonoid-dependent activation formed either no protein DNA complex or formed a complex with altered mobility compared to that obtained with extracts from wild-type strains.

Preferential DNA repair of an active gene in human cells.

Removal of pyrimidine dimers was measured in defined sequences in human cells amplified for the dihydrofolate reductase (DHFR) gene. We quantitated repair in specific restriction fragments by using the dimer-specific bacteriophage T4 endonuclease V and analysis by Southern blotting. Within 4 hr after 5- or 10-J/m2 UV irradiation, more than 60% of the dimers had been removed from a 20-kilobase fragment that lies entirely within the transcription unit of the DHFR gene and from a 25-kilobase fragment located in the 5' flanking region of the gene. Repair in the overall genome was measured by analyzing cellular DNA treated with T4 endonuclease V in alkaline sucrose gradients. Sixty-nine percent of the dimers were removed from the genome overall within 24 hr after irradiation, but only 25% were removed within 4 hr and 38% were removed within 8 hr. These results demonstrate a strong preferential rate of removal of dimers from the 50-kilobase region that includes the transcriptionally active DHFR gene compared to that in total cellular DNA. We confirmed that DHFR-containing DNA is repaired more rapidly than bulk DNA by using an approach that provides a direct comparison between repair in specific sequences and repair in total cellular DNA. We also show that the DHFR-containing sequences are repaired more rapidly than the nontranscribed repetitive alpha DNA sequences. Our finding of preferential early repair in a transcriptionally active region in overall repair-proficient cells suggests that selective dimer removal from active sequences may be a general characteristic of mammalian DNA repair.

Transcription unit of the chicken histone H5 gene and mapping of H5 pre-mRNA sequences.

We have analyzed the transcription unit of the gene coding for the erythrocyte-specific histone H5. RNA transcripts elongated in vitro by permeabilized immature cells hybridized to the template strand of the structural gene as well as to 3'-flanking sequences. Approximately 90% of the engaged RNA polymerase II molecules terminate transcription within a region of about 500 base pairs immediately downstream of the polyadenylation site. S1 nuclease protection experiments indicated that the downstream sequences are also transcribed in vivo, their relative amounts reflecting the distribution of RNA polymerases observed in vitro. RNA molecules extending up to 1.14 kilobase pairs downstream of the polyadenylation site were detected, but no unique site of termination was found. The sequence of the transcription termination region shows no obvious homology to those of other RNA polymerase II termination regions. The possible involvement of altered DNA and/or chromatin structures in the transcription termination process is discussed.

Transcription, processing and nuclear transport of a B1 Alu RNA species complementary to an intron of the murine α-fetoprotein gene

The Alu sequence family comprises the major dispersed repeat sequences of rodent and primate genomes, numbering greater than 300,000 copies in the human haploid genome. The function of these elements is unknown. The sequences can be transcribed by RNA polymerase III and represent a substantial fraction of total heterogeneous nuclear RNA. Alu sequences can be found both in the flanking regions and within the transcription units of several well-characterized genes. Here we show that some members of the mouse B1 Alu sequence family encode a small cytoplasmic RNA. The mouse B1 sequence is congruent to 130 nucleotides long and shows homology with the monomeric units of the dimeric 300-nucleotide primate sequence. By means of microinjection studies in the Xenopus laevis oocyte, we have elucidated a novel pathway leading to the appearance of a processed B1-type Alu RNA species in the cytoplasm. The abundance of this small Alu RNA differs between various mouse tissues, suggesting a role in tissue-specific gene expression.

Two simultaneously active VSG gene transcription units in a single trypanosoma brucei variant

Trypanosomes can change their surface coat either by slotting a different surface antigen gene copy into an active (telomeric) expression site or by activating a new VSG gene expression site and inactivating the old one. How expression sites are activated or inactivated is not clear. We report an exceptional trypanosome variant in which the inactivation of a surface antigen gene is accompanied by a 30 kb DNA insertion 5' of the gene. Transcription of the region upstream of the insertion continues unaltered and retains the characteristic insensitivity to alpha-amanitin of VSG gene transcription units, showing that the expression site is still active. The expressed VSG gene in this trypanosome variant resides in another telomere. Hence, two VSG gene transcription units can be simultaneously active. This argues against a single mobile activating element controlling VSG gene transcription and favors a stochastic model of telomere activation/inactivation.

Structure and transcription of a telomeric surface antigen gene of Trypanosoma brucei.

The gene encoding variant surface glycoprotein 221 in Trypanosoma brucei is located adjacent to a chromosome end and can be activated with or without a concomitant gene duplication. To test whether transcription initiates within the cloned segment of the 221 gene, we analyzed nascent and stable transcripts. We show here that the 221 coding region and 8.5 kilobases of adjacent upstream DNA are transcribed into nascent RNA at a similar rate when gene 221 is activated without duplication. Since only part of this transcribed upstream segment is transferred with the coding region to another telomere upon duplicative activation of gene 221, we infer that initiation of variant surface glycoprotein gene transcription occurs outside the gene segment that moves into an expression site by gene conversion. Our analysis shows that part of the variant surface glycoprotein 221 transcription unit consists of an unusual 3.5-kilobase tandem array of ca. 50 repeat segments and that a rearrangement in this array accompanies the nonduplicative activation of gene 221. A variant surface glycoprotein pseudogene is located within the transcription unit of gene 221, and we discuss models that account for this unusual situation.

Structure and transcription of a telomeric surface antigen gene of Trypanosoma brucei.

The gene encoding variant surface glycoprotein 221 in Trypanosoma brucei is located adjacent to a chromosome end and can be activated with or without a concomitant gene duplication. To test whether transcription initiates within the cloned segment of the 221 gene, we analyzed nascent and stable transcripts. We show here that the 221 coding region and 8.5 kilobases of adjacent upstream DNA are transcribed into nascent RNA at a similar rate when gene 221 is activated without duplication. Since only part of this transcribed upstream segment is transferred with the coding region to another telomere upon duplicative activation of gene 221, we infer that initiation of variant surface glycoprotein gene transcription occurs outside the gene segment that moves into an expression site by gene conversion. Our analysis shows that part of the variant surface glycoprotein 221 transcription unit consists of an unusual 3.5-kilobase tandem array of ca. 50 repeat segments and that a rearrangement in this array accompanies the nonduplicative activation of gene 221. A variant surface glycoprotein pseudogene is located within the transcription unit of gene 221, and we discuss models that account for this unusual situation.

Transcription Unit of the Rabbit β1 Globin Gene

We have hybridized pulse-labeled nuclear transcripts to cloned DNA fragments from the rabbit beta-like globin genes to determine the developmental timing, extent, and asymmetry of their transcription. The fetal-adult gene beta 1 was transcribed in fetal liver but not embryonic nuclei, whereas genes beta 3 and beta 4, which encode embryonic globin polypeptides, were transcribed only in embryonic nuclei. This shows that the switch from embryonic to fetal-adult globin production in rabbits is accomplished primarily by differential transcription of the beta-like globin genes. Gene beta 1 was subdivided into M13 subclones and tested for hybridization to nascent RNA. The nucleotide sequence of the 3' flanking region of gene beta 1 was also determined for 2,447 base pairs past the polyadenylation [poly(A)] site. No transcripts were found 5' to the cap site, but asymmetric transcription of gene beta 1 proceeded at a high level through the gene and past the poly(A) addition site for 603 nucleotides. The level of transcription declined after this, gradually dropping through the next 568 nucleotides. No polymerases were found on a fragment that begins 1,707 nucleotides past the poly(A) site; this fragment was part of a segment of repetitive DNA. These data show that the transcription unit of gene beta 1 begins at or near the cap nucleotide and extends at least 1,171 but no more than 1,706 nucleotides past the poly(A) addition site. The DNA segment that precedes the region of declining transcription contained an inverted repeat and encoded a short RNA transcribed by RNA polymerase II from the strand opposite the beta 1 transcript. These two features may function to attenuate the transcription of gene beta 1. An inverted repeat and a potential polymerase II transcription unit were also found in the homologous segment 3' to the human beta-globin gene. A short DNA segment close to the 3' end of the beta 1 transcription unit was transcribed more actively than the surrounding DNA, and it contained sequences that match the consensus internal control region for RNA polymerase III. This DNA segment may contain a separate polymerase III transcription unit. A member of the D repeat family located 3' to gene beta 1 was not transcribed in its entirety coordinately with beta 1.

Structure and polymorphism of class I MHC antigen mRNA.

We have used cDNA cloning and primer extension techniques to determine the complete nucleotide sequence of HLA-B7 mRNA. The 5'-untranslated sequence of the mRNA is rather short and the putative promoter has weak homology to the conventional "TATA" sequences. The 5' end and the polyadenylation site define the transcription unit of the B7 gene to be about 3.5 kb long. Comparison of the translated nucleotide sequence of this cDNA with the amino acid sequence of the heavy chain of the B7 antigen showed two amino acid differences. In addition, comparison with the sequences of the coding and untranslated regions of several HLA and H-2 genes showed that the class I histocompatibility molecules consist of four variable segments separated by three regions of homology. Analysis of the DNA polymorphisms revealed that in the variable segments the majority of the nucleotide substitutions are nonsilent, while in the homology regions the majority of substitutions are silent. Further analysis of the nature of the amino acid substitutions revealed the predominance of nonconservative replacements/changes in both the variable segments and the homology regions except the transmembrane part of the molecule. Selection at both the protein and the codon levels contributes to the pattern of mutations found in class I histocompatibility molecules.