Kb Of Upstream Sequence Research Articles

Spatial Pattern of Expression of Cyl Actin-β-Galactosidase Fusion Genes injected into Sea Urchin Eggs.

The Cyl actin gene of the sea urchin Strongylocentrotus purpuratus displays a pattern of expression that is correlated with cell division; the gene is initially activated in all cells of the early blastula stage embryo, but after 18 hours Cyl actin transcripts disappear from the aboral ectoderm at a time when these cells are withdrawing from the cell cycle. As part of our investigation of the transcriptional regulation of Cyl, we tested various Cyl-β-gal fusion genes for their spatial pattern of expression by microinjection into fertilized eggs of the sea urchin, Lytechinus pictus. The plasmid Cyl-β-gal containing 2.5 kb of upstream sequence displayed a pattern of expression which reflected the endogenous Cyl actin gene. Most of the staining was seen in the gut, oral ectoderm, and mesenchyme cells, while fewer embryos had stained cells in the aboral ectoderm cells. Deletions in the upstream sequences to-195 bp resulted in a decrease in the mean number of stained cells, but the overall pattern of expression was similar to Cyl-β-gal. These results indicate that sequences required for correct spatial expression of Cyl are located in the first 195 bp upstream of the Cyl start of transcription and/or within the first intron. A mutation in the highly conserved serum response element and an adjacent protein binding site did not increase the percentage of aboral ectoderm staining nor decrease the relative level of CAT activity from a Cyl-CAT fusion gene containing the same mutation. The implications of these rsults are discussed.

Different regulation of class I gene expression in the adult mouse and during development.

The expression of the class I genes encoding for histocompatibility Ag is complex both in adult and during development. Although ubiquitously expressed in the adult, the mRNA level of class I genes is variable from one organ to another. During development, H-2K mRNA expression has two phases: the first from blastocyst to day 11, where H-2K mRNA level is extremely low, and the second, beginning after day 11, when H-2K mRNA expression increases first dramatically (10x) and then progressively to birth. To localize the sequences responsible for the regulation of H-2K gene expression in the adult and during development, we have constructed a series of transgenic strains carrying 1) a 9-kb native H-2K gene, H-2K LF, corresponding to the entire H-2Kb gene with 2 kb of upstream sequences and 3 kb of downstream sequences, and 2) two hybrid constructs linking the same 5'-flanking region of H-2Kb gene to two reporter genes, the human growth hormone and the human c-myc proto-oncogene. Expression of the transgenes was compared with that of the endogeneous H-2K gene in adult organs and during development of the different transgenic strains. In the adult, the three constructs behave almost like the endogeneous H-2K gene, but the H-2K LF construct is the only one whose expression is independent of the integration site and related to the copy number. During development, both fusion genes are barely expressed in the embryo as well as in the extra-embryonic tissues, whereas the H-2K LF transgene expression parallels that of the endogeneous class I gene. Therefore, our results show that H-2K developmental regulatory sequences are not included in the region that controls H-2K mRNA expression in the adult, indicating that H-2K class I gene expression in adult organs and in development is regulated by different mechanisms.

Analysis of the promoter for the gene encoding the testis-specific histone H1t in a somatic cell line: evidence for cell-cycle regulation and modulation by distant upstream sequences

Gene H1t encodes a testis-specific variant of the H1 histone family expressed in pachytene spermatocytes during the meitotic phase of spermatogenesis. Fusions between various upstream fragments of the Hlt gene and the chloramphenicol acetyltransferase-encoding reporter gene analyzed in mouse L cells by both transient and permanent transfection. Expression of the minimal promoter [174 nucleotides (nt) upstream from the transcription start point] was enhanced by sequencesextending to nt −693, but was reduced in constructs with kb of upstream sequence. Using synchronized cells, expression was at least twofold higher in growing than in inhibited cells. Thus, the Hlt promoter is modulated both positively and negatively by distant upstream sequences, and it displays some S-phase-dependent character of a replication-dependent histone.

Primary structure of a novel barley gene differentially expressed in immature aleurone layers

As a direct approach to elucidate the molecular biology of barley aleurone cell development, we differentially screened an aleurone cDNA library made from poly(A)+ RNA of immature grains for clones representing transcripts present in the aleurone but not in the starchy endosperm. For one of these clones, B22E, which hybridies to a 0.7 kb transcript, Northern and in situ hybridization revealed that expression is under complex spatial, temporal and hormonal control in barley grains. cDNAs corresponding to B22E transcripts were isolated from aleurone/pericarp and embryo of developing grains, and from germinating scutella. Among these were the nearly full-length aleurone/pericarp clone pB22E.a16 (541 bp). cDNAs matching the sequence of this clone (type 1 transcript) were found for all tissues investigated. In addition, cDNAs with an extra 12 bp insertion (type 2 transcript) were obtained from germinating scutella. The two different transcripts can encode novel barley proteins of 115 and 119 amino acids, respectively. A gene designated B22EL8 was isolated and sequenced; it encodes the type 1 B22E transcript and contains two introns of 145 and 125 bp. Particle bombardment of barley aleurone with a B22EL8 promoter-GUS (beta-glucuronidase) construct demonstrates that the promoter (3 kb) is active in developing barley grains. The promoter is not, however, active in the seeds of tobacco plants transgenic for the B22EL8 gene, indicating the existence of sequences specific for monocots. A comparison of 1.4 kb of upstream sequence of B22E with the maize c1 promoter reveals a number of short, identical sequences which may be responsible for aleurone cell-specific gene transcription.

Upstream sequences regulating legumin gene expression in heterologous transgenic plants

We have previously isolated a legumin gene LeB4 from Vicia faba and shown that a 4.7 kb DNA fragment containing the gene leads to seed-specific expression in transgenic tobacco plants. Here we report that the 2.4 kb upstream sequence alone, when fused to either the neomycin phosphotransferase II (nptII) gene or the beta-glucuronidase (uidA) gene, leads to high enzyme levels in transgenic seeds of both tobacco and Arabidopsis. beta-Glucuronidase (GUS) activity is especially intense in the cotyledons fading out towards the embryonal root tip, a result confirmed by in situ hybridization. Staining of endosperm cells is consistent in both species. Analysis of a series of promoter deletion mutants fused to the nptII gene and introduced into tobacco plants revealed that about 1 kb of 5'-flanking sequence is sufficient for high-level expression but indirect evidence suggests the presence of weak positive regulatory elements further upstream. Deletions leaving only 0.2 kb of upstream sequence reduce enzyme levels to less than 10%. A deletion which destroys the legumin box with its seed protein gene-specific CATGCATG motif has no obvious effects on expression levels.

Evidence for positive and negative regulation of the Hox-3.1 gene.

The region-specific patterns of expression of mouse homeobox genes are considered important for establishing the embryonic body plan. A 5-kilobase (kb) DNA fragment from the Hox-3.1 locus that is sufficient to confer region-specific expression to a beta-galactosidase reporter gene in transgenic mouse embryos has been defined. The observed reporter gene expression pattern closely parallels endogenous Hox-3.1 expression in 8- to 9.5-day postcoitum (p.c.) embryos. At 10.5 days p.c. and later, the pattern of beta-galactosidase activity diverges from the Hox-3.1 pattern, and an inappropriately high level of reporter gene expression is observed in posterior spinal ganglia. Inclusion of an additional 2 kb of upstream sequences is sufficient to suppress this aberrant expression in the developing spinal ganglia. Together, these results show that the control of early Hox-3.1 expression is complex, involving at least one positively acting and one negatively acting element.

Repetitive sequence involvement in the duplication and divergence of mouse lysozyme genes.

Mouse M and P lysozymes are the products of separate genes, are specifically expressed in separate tissues, and are adapted to different functions. The lysozyme genes have assumed these markedly different characteristics following their generation by gene duplication 30-50 million years ago. The discovery of the lysozyme P gene only 5 kb upstream from the M gene in tandem repeat has enabled an investigation of the molecular basis of their duplication and subsequent divergence. The duplication is shown to have involved recombination between two B2 repeat sequences flanking the original gene. The resulting downstream copy has retained the myeloid specificity of expression along with just 1.7 kb of upstream sequences, while the upstream copy is inactive in macrophages and has become expressed instead in the small intestine. Although multiple gene conversion events have served to maintain a generally high homology between the genes, certain regions have been found to be specific for either one of the gene pair: two repetitive sequences peculiar to the P region may serve to protect the coding regions from gene conversion, while sequences unique to the M gene may be more directly involved in differential regulation.

Purine Synthesis de novo and Salvage in Hypoxanthine Phosphoribosyltransferase-Deficient Mice

Extreme degrees of hypoxanthine phosphoribosyltransferase (HPRT) deficiency in man are associated with gross sex-linked neurological dysfunction, gout and urinary stones (the Lesch-Nyhan or 'complete HPRT-deficiency' syndrome). The less severe degrees of enzyme deficiency (sex-linked recessive gout and/or urolithiasis or the 'partial HPRT-deficiency' syndrome) may be associated with minor neurological manifestations. Whole body purine synthesis de novo is accelerated in both these groups of patients. A strain of mice with an experimentally produced mutation at the HPRT locus showed some residual 'apparent HPRT activity' in brain, liver, testicular, splenic, kidney and ovarian tissues but not in erythrocyte haemolysates. The mutation removes exons 1 and 2 of the coding region of the gene together with the promotor and about 10 kb of upstream sequence from the gene. It is therefore possible that the observed 'apparent HPRT activity' in these mice is due to the operation of an alternative metabolic pathway. Purine synthesis de novo was markedly accelerated in their brain, testicular, splenic and kidney tissues. It was not accelerated in the liver tissue of male mice hemizygous for the mutation and the degree of acceleration in the female homozygotes only just reached statistical significance at the p = 0.02 level. This observation casts doubt on the importance of modulations in the rate of hepatic purine synthesis de novo as a mechanism for maintaining a steady supply of purines for translocation to other organs.

Structure of the human retinoblastoma gene.

Complete inactivation of the human retinoblastoma gene (RB) is believed to be an essential step in tumorigenesis of several different cancers. To provide a framework for understanding inactivation mechanisms, the structure of RB was delineated. The RB transcript is encoded in 27 exons dispersed over about 200 kilobases (kb) of genomic DNA. The length of individual exons ranges from 31 to 1889 base pairs (bp). The largest intron spans greater than 60 kb and the smallest one has only 80 bp. Deletion of exons 13-17 is frequently observed in various types of tumors, including retinoblastoma, breast cancer, and osteosarcoma, and the presence of a potential "hot spot" for recombination in the region is predicted. A putative "leucine-zipper" motif is exclusively encoded by exon 20. The detailed RB structure presented here should prove useful in defining potential functional domains of its encoded protein. Transcription of RB is initiated at multiple positions and the sequences surrounding the initiation sites have a high G + C content. A typical upstream TATA box is not present. Localization of the RB promoter region was accomplished by utilizing a heterologous expression system containing a bacterial chloramphenicol acetyltransferase gene. Deletion analysis revealed that a region as small as 70 bp is sufficient for RB promoter activity, similar to other previously characterized G + C-rich gene promoters. Several direct repeats and possible stem-and-loop structures are found in the promoter region. No enhancer element was detected within the 7.3 kb of upstream sequence studied. Several features of the RB promoter are reminiscent of the characteristics associated with many "housekeeping" genes, consistent with its ubiquitous expression pattern.

Purine synthesis de novo and salvage in hypoxanthine phosphoribosyltransferase-deficient mice

Extreme degrees of hypoxanthine phosphoribosyltransferase (HPRT) deficiency in man are associated with gross sex-linked neurological dysfunction, gout and urinary stones (the Lesch-Nyhan or 'complete HPRT-deficiency' syndrome). The less severe degrees of enzyme deficiency (sex-linked recessive gout and/or urolithiasis or the 'partial HPRT-deficiency' syndrome) may be associated with minor neurological manifestations. Whole body purine synthesis de novo is accelerated in both these groups of patients. A strain of mice with an experimentally produced mutation at the HPRT locus showed some residual 'apparent HPRT activity' in brain, liver, testicular, splenic, kidney and ovarian tissues but not in erythrocyte haemolysates. The mutation removes exons 1 and 2 of the coding region of the gene together with the promotor and about 10 kb of upstream sequence from the gene. It is therefore possible that the observed 'apparent HPRT activity' in these mice is due to the operation of an alternative metabolic pathway. Purine synthesis de novo was markedly accelerated in their brain, testicular, splenic and kidney tissues. It was not accelerated in the liver tissue of male mice hemizygous for the mutation and the degree of acceleration in the female homozygotes only just reached statistical significance at the p = 0.02 level. This observation casts doubt on the importance of modulations in the rate of hepatic purine synthesis de novo as a mechanism for maintaining a steady supply of purines for translocation to other organs.

Structure of the rabbit fast-twitch skeletal muscle Ca2+-ATPase gene.

We have isolated two genomic clones which together encode the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. One of the two 16.5 kilobase (kb) genomic inserts in the lambda phage vector Charon 4A contains 23 exons extending from the polyadenylation site at the 3' end of the ATPase gene to within 38 nucleotides of the translation initiation codon in the 5' exon. An overlapping genomic insert of 16.5 kb contains the remainder of the 5' exon and a further 8 kb of upstream sequence. S1 nuclease mapping and primer extension analysis of the 5' end of the Ca2+-ATPase mRNA indicate that the transcription initiation site is located 185 base pairs (bp) upstream of the translation initiation codon. A "TATA" box (CA-TAAA) was found at position -30 and the sequence CCAAT was found at position -78 relative to the transcription initiation site. In a previous study (Brandl, C. J., de Leon, S., Martin, D. R., and MacLennan, D. H. (1987) J. Biol. Chem. 262, 3768-3774) cDNAs for neonatal and adult forms of the fast-twitch Ca2+-ATPase were shown to encode different carboxyl-terminal sequences, presumably as a result of alternative splicing. We have now found that these different DNA sequences encoding different carboxyl-terminal sequences are located in different exons. Exon boundaries of the Ca2+-ATPase gene did not correlate well with proposed domain boundaries for the Ca2+-ATPase protein. The locations of exon/intron boundaries were only partially conserved between the Ca2+-ATPase gene and a Na+/K+-ATPase gene (Ovchinnikov, Y. A., Monastyrskaya, G. S., Broude, N. E., Allikmets, R. L., Ushkaryov, Y. A., Melkov, A. M., Smirnov, Y. V., Malyshev, I. V., Dulubova, I. E., Petrukhin, K. E., Gryshin, A. V., Sverdlov, V. E., Kiyatkin, N. I., Kostina, M. B., Modyanov, N. N., and Sverdlov, E. D. (1987) FEBS Lett. 213, 73-80) and they did not follow closely the boundaries of amino acid sequences that are highly conserved among a group of ion transport ATPases.

Molecular characterization of spalt, a homeotic gene required for head and tail development in the Drosophila embryo

The isolation, identification and structure of the spalt gene is described. This novel homeotic gene of Drosophila is required for the establishment of the posterior-most head and the anterior-most tail segments of the embryo. It encodes a small mRNA of 0.8 kb which is under the control of over 15 kb of upstream sequences as indicated by the phenotype of transformed embryos. The putative spalt protein contains internal repeats and other interesting structural motifs but no homeo box. The spalt transcript accumulates motifs but no homeo box. The spalt transcript accumulates to high levels in the segmental anlagen affected in mutant embryos but is also found in regions of the embryo where no functional requirement has been demonstrated.

Transformation of salivary gland secretion protein gene Sgs-4 in Drosophila: stage- and tissue-specific regulation, dosage compensation, and position effect.

The Sgs-4 gene of Drosophila melanogaster encodes one of the larval secretion proteins and is active only in salivary glands at the end of larval development. This gene lies in the X chromosome and is controlled by dosage compensation--i.e., the gene is hyperexpressed in males. Therefore, males with one X chromosome produce nearly as much Sgs-4 products as females with two X chromosomes. We used a 4.9-kilobase-pair (kb) DNA fragment containing the Sgs-4d coding region embedded in 2.6 kb of upstream sequences and 1.3 kb of downstream sequences for P-element-mediated transformation of the Sgs-4h underproducer strain Kochi-R. Sgs-4d gene expression was found in all 15 transformed lines analyzed, varying with the site of chromosomal integration. The transposed gene was subject to tissue- and stage-specific regulation. At X-chromosomal sites, the levels of gene expression were similar in both sexes, signifying dosage compensation. At autosomal sites, it was on average 1.5 times higher in males than in females. The results indicate that the transforming DNA fragment contains all sequences necessary for tissue- and stage-specific regulation and for hyperexpression in males.

Steroid controlled expression of the chicken lysozyme and the rat tryptophan oxygenase gene after transfer into eukaryotic cells

To study the mechanism of steroid induced transcriptional control we have introduced recombinants of the chicken lysozyme gene and the rat tryptophan oxygenase (TO) gene into heterologous and homologous cells. To monitor the activity of the TO-promoter, 1.9 kb of the TO 5'-flanking sequences were fused with sequences coding for the bacterial enzyme chloramphenicol acetyltransferase (CAT). Upon transfer into mouse l-cells the transient expression of the TO-CAT recombinant was found to be inducible by dexamethasone. Transient expression of chicken lysozyme gene recombinants after introduction into various cell types could only be detected in chicken oviduct cells, or in conjunction with SV 40 enhancer sequences in human cells. The recombinant gene used in oviduct cells was a fusion between the lysozyme promoter, including 1.4 kb of upstream sequences, and the coding region of the gene for SV 40 T-antigen (plys-T). The expression in oviduct cells was stimulated by dexamethasone or progesterone, whereas SV 40 enhanced expression of lysozyme sequences in human cells could not be regulated by steroids. Using several deletion mutants, a region between −220 bp and −140 bp upstream of the cap site was found to be essential for both regulation by glucocorticoids as well as by progesterone.

Expression of a chicken lysozyme recombinant gene is regulated by progesterone and dexamethasone after microinjection into oviduct cells

We transferred a chicken lysozyme gene recombinant by microinjection into primary cultures of chicken oviduct cells. The recombinant gene is a fusion between the lysozyme promoter, including 1.4 kb of upstream sequences, and the coding region of the gene for SV40 T antigen (plys-T). The expression of plys-T is stimulated by the steroid hormones progesterone and dexamethasone, but not by estradiol. The number of oviduct cells expressing coinjected or separately injected control genes is not increased by steroids. A deletion mutant lacking the lysozyme sequences between −161 and +15 does not express T antigen, indicating that transcription of plys-T starts within the lysozyme promoter region. By screening different cell types we found that microinjected plys-T is expressed in chicken oviduct cells but not in chicken macrophages or fibroblasts or in rat II fibroblasts.