Transcription Unit Of Gene Research Articles

Sequence of murine CDC10 cDNA, gene organization and expression analysis

The sequence of the 2391 bp murine CDC10 cDNA is reported. The gene transcription unit, composed of 13 exons, encodes a potential 417/446 amino acid GTP-binding protein, highly similar to human CDC10. The ubiquitous expression of the gene, as judged by Northern analysis, is consistent with its putative promoter structure.

A 33 bp minisatellite repeat upstream of the 'mutated in colon cancer' gene at chromosome 5q21.

We have found a 33 bp minisatellite repeat in the 5'-flanking region of the mutated in colon cancer (MCC) gene at chromosome 5q21. Southern blot experiments demonstrated the locus specificity of the repeat. The number of repeat units varied between 5 and 11 with a heterozygosity of 0.56. The sequence 5'-AGG AGT GTG AAT GGG GCA TAG TGA ATG AGG GGA-3' of the repeat units does not match the consensus sequence of chi-related minisatellites. The minisatellite is not expressed as part of a gene transcription unit. However, it can be used as a tool for the detection of allelic changes at chromosome 5q21 on standard agarose gels.

Cloning and Expression of a Novel Gene Encoding a New Antibacterial Peptide from Silkworm,Bombyx mori

We differentially screened a novel gene encoding a new antibacterial peptide from the immunizedBombyx moricDNA library. The gene showed a similar structure to that of cecropin- family, encoding 59 amino acids including a putative leader peptide and mature peptide. The deduced peptide, named Enbocin, had conserved amino acid residues which have been known to play an important role in the antibacterial activities. Enbocin genomic sequence revealed that the transcription unit of Enbocin gene was about 1.2 kb, and the coding sequence was interupted by an intron of 660 bases. Recombinant Enbocin, expressed under the control of the baculovirus polyhedrin promoter, demonstrated a broad range of antibacterial activities against gram positive and gram negative bacteria.

Characterization and Retinoic Acid Responsiveness of the Murine Hoxd4 Transcription Unit

We have characterized the transcription unit of a murine Hox gene in the fourth paralogous group, Hoxd4. We have identified two Hoxd4 transcription start sites by S1 analysis. The upstream promoter (P2) is 5.2 kilobase pairs upstream from the coding region, while the downstream promoter (P1) is 1.1 kilobase pairs distant. Both promoters bear a cluster of start sites. Multiple transcripts were identified by Northern blot, originating from both promoters and multiple polyadenylation signals. Expression of P1 transcripts in the neural tube shows an anterior border at the rhombomere 6/7 boundary, corresponding to previous reports (Gaunt, S. J., Krumlauf, R., and Duboule, D. (1989) Development 107, 131-141; Morrison, A., Moroni, M. C., Ariza-McNaughton, L., Krumlauf, R., and Mavilio, F. (1996) Development 122, 1895-1907). A more posterior boundary in the central nervous system was observed for P2 transcripts. We observed strong expression up to somite 6 and weak expression in somite 5, correlating with the phenotype of Hoxd4 null mutant mice (Horan, G. S. B., Nagy Kovàcs, E., Behringer, R. R., and Featherstone, M. S. (1995) Dev. Biol. 169, 359-372). In response to retinoic acid, expression from P1 in the hindbrain was anteriorized after 4 or 24 h of treatment. P2 transcripts seemed to be less responsive and/or to have an indirect response to retinoic acid. The long 5'-untranslated region found in all Hoxd4 transcripts suggests that translation does not occur by a classical ribosome scanning mechanism.

The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex.

Multiple members of the dynein heavy chain (Dhc) gene family have been recovered in several organisms, but the relationships between these sequences and the Dhc isoforms that they encode are largely unknown. To identify Dhc loci and determine the specific functions of the individual Dhc isoforms, we have screened a collection of motility mutants generated by insertional mutagenesis in Chlamydomonas. In this report, we characterize one strain, pf9-3, in which the insertion event was accompanied by a deletion of approximately 13 kb of genomic DNA within the transcription unit of the Dhc1 gene. Northern blot analysis confirms that pf9-3 is a null mutation. Biochemical and structural studies of isolated axonemes demonstrate that the pf9-3 mutant fails to assemble the I1 inner arm complex, a two-headed dynein isoform composed of two Dhcs (1 alpha and 1 beta) and three intermediate chains. To determine if the Dhc1 gene product corresponds to one of the Dhcs of the I1 complex, antibodies were generated against a Dhc1-specific peptide sequence. Immunoblot analysis reveals that the Dhc1 gene encodes the 1 alpha Dhc subunit. These studies thus, identify the first inner arm Dhc locus to be described in any organism and further demonstrate that the 1 alpha Dhc subunit plays an essential role in the assembly of the I1 inner arm complex.

Expression of caprine beta-lactoglobulin in the milk of transgenic mice.

A 14.5 kb-long transgene containing the complete caprine beta-lactoglobulin gene transcription unit as well as 6.1 kb and 3.7 kb of the 5'- and 3'-flanking regions, respectively, was microinjected into pronuclear stage mouse embryos. Four lines of transgenic mice were obtained, three of them expressing the transgene in their mammary glands during lactation. Western blot analysis of caprine beta-lactoglobulin in the milk of hemizygous transgenic animals demonstrated the presence of the exogenous protein at concentrations up to 0.5 mg ml-1 of mouse milk.

Characterization and localization of the mProx1 gene directly upstream of the mouse alpha-globin gene cluster: identification of a polymorphic direct repeat in the 5'UTR.

The alpha-globin major regulatory element (alpha MRE) positioned far upstream of the gene cluster is essential for the proper expression of the alpha-globin genes. Analysis of the human and mouse alpha-globin Upstream Flanking Regions (alpha UFR) has identified three nonglobin genes in the order Dist1-MPG-Prox1-alpha-globin. Further characterization of the whole region indicates that the alpha MRE and several other erythroid DNase HSSs are associated with the transcription unit of the Prox1 gene. In this paper we describe the characterization and localization of the mouse Prox1 cDNA and compare it with its human homolog, the -14 gene, and another human cDNA sequence named hProx1. Our results show a strong conservation between the -14 gene and the mouse Prox1 gene with the exception of the first exon of the mProx1 gene. This exon is absent in the -14 cDNA but is present and conserved in the human Prox1 cDNA, indicating that the human -14/hProx1 gene is alternatively spliced or transcribed. The mProx1 gene encodes a predicted protein of 491 amino acids (aa) whose function is not known. In the 5'UTR of this gene, a 35-bp repeat (VNTR) is positioned, which is highly polymorphic among laboratory inbred mice (Mus domesticus). Our results strongly suggest that the mProx1 VNTR arose during the divergence of M. spretus and M. domesticus. Besides its use in evolutionary studies and positional cloning, the mProx1 VNTR might be invaluable for monitoring the expression of a transgenic mProx1 gene. The cloning of the mProx1 gene will be helpful to analyze its possible role on alpha-globin as well on MPG expression in the mouse.

Expression of c-myc gene and biosynthesis of biological macromolecules in antisense transfectant HL60 R-9

The recombinant plasmid PGC was constructed for transcription unit of c-myc gene with diorientationin vitro, to make RNA probes for detection of c-myc mRNA and antisense RNA expression of transfectant HL R60 -9, which was obtained from HL60 cells transfected with inducible c-myc antisense RNA expression plasmid. The results from HL R60 -9 cells induced by Cd2+ indicated that expression of c-myc antisense RNA increased with Cd2+ concentration and exposure time, while c-myc mRNA expression progressively reduced. Using immunohistochemical technique no c-myc P62 protein expression was detected. The incorporation of3H-TdR,3H-UR and3H-Leu revealed significant suppression of DNA, RNA and protein biosynthesis. It is suggested that the reversion changes previously reported in malignant phenotypes of HL R60 -9 cells and the inhibition of macromolecular biosynthesis mentioned above were associated with the blockade of c-myc gene expression by its antisense RNA.

Molecular characterization of an 18 kb segment of DNA puff C4 of Bradysia hygida (Diptera, sciaridae).

The data presented here are an extension of the molecular characterization of DNA puff C4 of Bradysia hygida. A cDNA related to a gene amplified in this puff and expressed when puff C4 expands was cloned and sequenced. Analysis of the amino acid sequence deduced from the open reading frame present in the cDNA indicate that the encoded protein is secreted and comprises mostly alpha-helical coiled-coil. An 18 kb genomic segment containing the transcription unit of this gene was also cloned and the structure and expression of the 1.4 kb mRNA was determined. Quantitative slot blot hybridization of DNA complementary to the transcription unit shows that this gene is amplified about 21 times in the salivary gland, confirming data previously obtained. Fragments upstream of the 5' end, and beyond the 3' end, of the gene transcription unit were also analysed and shown to be amplified at least eight and five times, respectively. Based on these data we discuss how amplification could occur at DNA puffs.

Promoter structure and intron-exon organization of a scorpion alpha-toxin gene.

The Androctonus australis scorpion venom contains alpha-toxins for which the complementary DNAs have been cloned [Bougis et al. (1989) J. Biol. Chem. 264, 19259-19265], targeting with high affinity the voltage-sensitive sodium channel. From a genomic library made of this species of scorpion, we have cloned and characterized the gene encoding the toxin AaH I'. The gene transcriptional unit is 793 base pairs long, and the gene has a single intron of 425 base pairs located near the end of the signal peptide of the toxin precursor. The transcription initiation site was determined by primer extension and corresponded to the nucleotide sequence AACAA. Upstream, a promoter region has been identified with positive acting sequence elements at consensus positions, such as a CCAAT box and a TATA box. In addition, putative elements for binding the transcriptional factors MAT-alpha 2, Pit-1, and IEF1 are also present. Analysis of DNA curvature by computer modeling revealed a strong bending centered around the transcription initiation site of the gene. The bending angle (61 degrees) estimated experimentally using polyacrylamide gel electrophoresis correlates well with the value predicted by computer modeling (66 degrees). Other minor deflections of the helix axis cooperate for an overall curvature of nearly 90 degrees, which is significantly stronger than similar structures already reported in eukaryotic cells. It is worth noting that the grooves relative to the CCAAT box and the TATA box lie along the inside of the DNA curve. This observation is in agreement with the previously reported correlation between DNA bending and promoter function.

Gamete–specific methylation correlates with imprinting of the murine Xist gene

We have investigated the potential role of DNA methylation as a regulator of imprinted Xist expression in mouse preimplantation embryos. The active paternal allele was found to be unmodified in sperm at CpG loci near the 5' end of the gene transcription unit. In contrast, on the inactive maternal allele, these same sites are initially methylated in the oocyte and then remain modified in the early embryo. In the male germ line, these methyl moieties are removed during spermatogenesis, and this occurs before the programmed reactivation of Xist in the testis. This represents a clear-cut example of a potential methylation imprinting signal that is reprogrammable and gamete derived.

The programmed cell death of an immature thymocyte cell line transgenic for an alpha beta TCR and the c-myc proto-oncogene.

The c-myc proto-oncogene linked to the mouse Thy-1 gene transcriptional unit predisposes mice to development of thymic tumors consisting predominantly of immature CD4+ CD8+ cells. In an attempt to immortalize immature T cells expressing a known T-cell antigen receptor (TCR), Thy-1/c-myc transgenic mice were bred to αβ TCR transgenic mice (F5), and CD4+ CD8+ cell lines were established from thymic tumors in double-transgenic mice. These cells expressed high-level heat-stable antigen (HSA) and were able to undergo programmed cell death upon induction with steroids and CD3 cross-linking, but not with cognate peptide. In addition, one line had rearranged and transcribed endogenous TCR c and genes, in spite of the fact that transgenic α and β genes were also expressed. Furthermore, we show that Thy-1/myc transgenic mice deficient in recombination activating gene-1 (RAG-1) do not develop tumors, in contrast to RAG-1-/- mice, which are also transgenic for both Thy-1/myc and the F5 TCR. This indicates that in order for thymocytes to be transformed by the Thy-myc transgene, they need to proceed to the double-positive stage.

Expression of a human insulin transgene in cholinergic neurons of the mouse medial habenula

We explored the possibility that an insulin gene deleted in its 5'-flanking region is expressed in adult mouse brain. We used three independent lines of mice carrying a human insulin transgene which included the insulin gene transcription unit flanked by 168 base pairs upstream and 5.5 kb downstream. Using a reverse transcription-polymerase chain reaction assay, human insulin mRNAs were detected in whole brain extracts. In all three lines, human insulin mRNAs were localized by in situ hybridization in a single cerebral site, the medial habenula. With a monoclonal antibody specific for human C-peptide and human proinsulin, labelling was restricted to a subset of habenular cholinergic neurons, with rare immunostained fibers. No labelling was observed in the projection fibers of the retroflexus fasciculus or in their axon terminals in the interpeduncular nucleus. Electron microscope studies suggested that the transgene expressing cells. These findings demonstrate that the human insulin transgene tested here includes a habenula specific promoter which could be useful for physiological and molecular studies on the habenula.

RNA Chain Elongation and Termination by Mammalian RNA Polymerase III: Analysis of tRNA Gene Transcription by Imposing a Reversible Factor-mediated Block to Elongation using a Sequence-specific DNA Binding Protein

We have used a sequence-specific DNA binding protein to examine transcription elongation and termination by mammalian RNA polymerase III (polIII). The Escherichia coli lac repressor protein, bound to its cognate operator site positioned between the 3′ end of the coding region and the termination site of a human tRNA gene, conditionally blocked transcription elongation by polIII in vitro in HeLa cell nuclear extracts. Arrest of elongation by polIII dramatically reduced overall levels of transcription and directed the synthesis of shortened transcripts, consistent with a block to polIII elongation at the boundary of the repressor/DNA complex. Removal of template-bound repressor with the allosteric inducer isopropylthio-β- d-galactoside (IPTG) allowed extension of nascent transcripts and restored transcriptional activity. Moreover, a subset of transcription complexes were shown to be capable of transcribing through the repressor obstacle lac repressor positioned just downstream of the natural termination site effected the premature termination of transcription but otherwise had no affect on the overall level of transcription. Our findings demonstrate that elongation and termination by mammalian polIII can be modulated in vitro by a heterologous sequence-specific DNA binding protein. Moreover, the ability to selectivity arrest elongation by polIII at defined positions within the tRNA gene transcription unit has permitted the identification of discrete functional properties of paused mammalian polIII ternary complexes.

Molecular Analysis of the T-Cell Acute Lymphoblastic Leukemia-Associated t(1;7)(p34;q34) That Fuses LCK and TCRB

Abstract Previously we had characterized the t(1;7)(p34;q34) translocation from HSB-2. This translocation fused the beta T-cell receptor gene (TCRB) constant region and transcriptional enhancer with the type I transcription unit of the LCK gene on the derivative 1 [der(1)] chromosome. The type II promoter was translocated to the der(7) chromosome. Regarding the mechanism of the t(1;7) in HSB-2, we identified an alternating purine-pyrimidine tract (G-T)17 at the 1p34/LCK breakpoint. Additionally, sequence analysis of both breakpoint junctions provided data that implicate the V(D)J recombinase in formation of the t(1;7). A heptamer-nonamer recognition sequence with a 12-bp spacer was found in the immediate vicinity of the 1p34/LCK breakpoint and, thus, chromosomal breakage at 1p34 may be explained as resulting from recombinase activity. Because phosphorylation of Tyr-505 in vivo regulates the tyrosine kinase activity of p56lck we amplified a region from LCK exon 12 that contains the codon for Tyr-505 and showed no mutation of this codon in HSB-2 DNA and, therefore, p56lck in HSB-2 is not activated by mutation of Tyr-505. We have analyzed LCK gene expression in HSB-2 and SUP-T12 cell lines. RNase protection analysis identified almost exclusively type I transcripts in HSB-2. An independent t(1;7) in SUP-T12 also resulted in the juxtaposition of LCK to TCRB. The breakpoint in SUP-T12 occurred 2 kb 5′ of the type II promoter, leaving an intact LCK gene on the der(1) chromosome. RNase protection analysis identified both type I and type II LCK transcripts in a 3:1 ratio in SUP-T12. Factors other than proximity to the TCRB enhancer must affect promoter utilization in this cell line.

Molecular analysis of the T-cell acute lymphoblastic leukemia- associated t(1;7)(p34;q34) that fuses LCK and TCRB

Previously we had characterized the t(1;7)(p34;q34) translocation from HSB-2. This translocation fused the beta T-cell receptor gene (TCRB) constant region and transcriptional enhancer with the type I transcription unit of the LCK gene on the derivative 1 [der(1)] chromosome. The type II promoter was translocated to the der(7) chromosome. Regarding the mechanism of the t(1;7) in HSB-2, we identified an alternating purine-pyrimidine tract (G-T)17 at the 1p34/LCK breakpoint. Additionally, sequence analysis of both breakpoint junctions provided data that implicate the V(D)J recombinase in formation of the t(1;7). A heptamer-nonamer recognition sequence with a 12-bp spacer was found in the immediate vicinity of the 1p34/LCK breakpoint and, thus, chromosomal breakage at 1p34 may be explained as resulting from recombinase activity. Because phosphorylation of Tyr-505 in vivo regulates the tyrosine kinase activity of p56lck we amplified a region from LCK exon 12 that contains the codon for Tyr-505 and showed no mutation of this codon in HSB-2 DNA and, therefore, p56lck in HSB-2 is not activated by mutation of Tyr-505. We have analyzed LCK gene expression in HSB-2 and SUP-T12 cell lines. RNase protection analysis identified almost exclusively type I transcripts in HSB-2. An independent t(1;7) in SUP-T12 also resulted in the juxtaposition of LCK to TCRB. The breakpoint in SUP-T12 occurred 2 kb 5' of the type II promoter, leaving an intact LCK gene on the der(1) chromosome. RNase protection analysis identified both type I and type II LCK transcripts in a 3:1 ratio in SUP-T12. Factors other than proximity to the TCRB enhancer must affect promoter utilization in this cell line.

Positive and negative regulatory elements are involved in transcriptional control of the rat glucokinase gene in the insulin producing cell line HIT M2.2.2

Nested deletion mutants of the 5' flanking region of the β-cell transcription unit of the rat glucokinase gene (rßGK) were fused to the CAT-reporter gene. Transient expression studies in HIT M2.2.2 and BHK21 cells revealed a distal (upstream of −359) and a proximal promoter region (between −278/−49) harbouring positive and negative regulatory elements. DNaseI footprinting revealed three protected areas between nucleotides −190 and −60. DNA-elements playing a crucial role in transcriptional control of the insulin genes (IEB- and CT-motifs) have been detected within the proximal promoter region and contribute to β-cell specific gene regulation. 3' deletion analysis revealed that DNA-elements located downstream from transcription initiation sites (up to +123) contribute to transcriptional regulation.

A new vector for the high level expression of chimeric antibodies in myeloma cells

We previously reported the expression of a mouse/human chimeric anti-ganglioside G D3 antibody, KM871 (IgG1,κ) in mouse myeloma SP2/0 cells under the control of the ecotropic Moloney virus long terminal repeat by the co-transfection of chimeric heavy (H) and light (L) chain vectors (Shitara et al. (1993) Cancer Immunol. Immunother.). To establish an efficient and high level expression system for the chimeric antibody, we did comparative study on vector systems and host cells. An improved expression vector, named ‘a tandem vector, pChi641HLGM4’ was constructed, in which both of the chimeric H and L chain gene transcription units and a dihydrofolate reductase (dhfr) gene transcription unit were inserted. When two kinds of mouse myeloma cell lines, SP2/0 and P3U1, were used as host cells, frequency of the incidence of antibody-producing transfectants was markedly increased by the use of the tandem vector compared with the use of the mixture of each chimeric H vector and L chain vector. To select out appropriate host cells, transfection frequency and antibody production level were compared among SP2/0, P3U1 and rat myeloma YB2/0 cells by transfection of the tandem vector. YB2/0 cell was shown to have the highest potential in both the transfection frequency and the antibody production. Introduction of the tandem vector into YB2/0 cells and the subsequent amplification with 50–200 nM methotrexate gave rise to several clones that stably secreted 70–100 μg/10 6 cells per 24 h of the chimeric antibody. This productivity of the antibody is one of the highest levels which have been achieved by other investigators using transfected myeloma cells. Using this system it took only 2–3 months to establish the transfectant clones which stably produced the chimeric antibody.

The role of the proximal CTAAT-box of the rat glucokinase upstream promoter in transcriptional control in insulin-producing cells.

Sequence analysis of the 5'flanking region of the beta-cell specific transcription unit of the rat glucokinase gene (r beta GK) revealed the presence of sequence motifs very similar to the IEB-(Far)-box and a CT-motif which play a crucial role in transcriptional control of insulin genes. 5'deletional analysis of the r beta GK proximal promoter element (localized between nucleotides -278 and -49) as well as site directed mutagenesis showed that both motifs are mutationally sensitive and contribute to transcriptional control in HIT M2.2.2 cells. The combination of the IEB-(Far)-like motif with the CT-box was unable to form a "mini-enhancer" similar to the Far-FLAT-element of the rat insulin I gene promoter but rather functions as a beta-cell specific control element in r beta GK expression. Electrophoretic mobility shift assays (EMSAs) and competition studies using oligonucleotides containing CT-motifs of rat insulin genes promoters, human insulin gene promoter, and rat amylin gene promoter showed similar binding patterns with nuclear extracts isolated from insulin-producing cell lines. These studies indicate that CT-motifs of rat glucokinase, insulin, and amylin gene promoters may bind similar--probably identical--nuclear factor(s) and may play a central role in the coordinated expression of these genes in insulin-producing cells.

Sequence of the intergenic spacer between the 28S and 18S rRNA-encoding genes of the crustacean, Daphnia pulex

The intergenic spacer ( IGS) of the ribosomal DNA of the crustacean, Daphnia pulex, is 4819 bp in length, and contains two non-repetitive regions separated by two repetitive regions. The larger repetitive region contains three complete and one truncated copy of a 330-bp sub-repeat. Each sub-repeat contains the sequence, 5'-TATATAGGGAAG, which is very similar to the promoter sequences found in repeated elements in the IGS of other organisms. The 3' non-repetitive region, which separates the repeat elements containing the putative transcription promoter sites from the 18S coding region, is large (2689 bp) compared to those described for other arthropods. A putative transcription start point ( tsp) has been identified 1280 bp upstream from the 18S coding region, suggesting that the external transcribed spacer is also large compared to that in other arthropods. The 3' non-repetitive region contains two poly(GT) tracts of 18 and 20 bp. Such sequences, which are capable of forming Z-DNA in vitro, have been found upstream from the transcription units of rRNA and protein-coding genes in human, mouse and rat, and are thought to have a functional role in gene expression.