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PubMed1984

Structure and comparison of the oxytocin and vasopressin genes from rat.

The gene encoding the precursor protein to the hormone oxytocin and its associated neurophysin has been isolated from a rat genomic library, and its sequence has been determined. The small gene (approximately equal to 850 base pairs) predicts a mRNA of approximately equal to 500 bases [without the poly(A) tail]. The exon-intron organization is similar to that of the vasopressin gene, with two splice sites in the protein-coding region. The first exon (A) comprises the 5' noncoding promoter region, a putative signal peptide, the nonapeptide hormone oxytocin, and the NH2-terminal, variable region of neurophysin. The second exon (B) encodes the central, conserved region of neurophysin, and the third exon (C) encodes the remaining COOH terminus of neurophysin, with an additional arginine residue at its end, presumably cleaved off during post-translational processing. A stretch of 143 nucleotides within exon B, except for a single base change, is entirely homologous to the equivalent part of the rat vasopressin gene, offering support for a gene conversion event having recently affected the two genes.

Animals↗

PubMed1993

C-ski transcripts with and without exon 2 are expressed in skeletal muscle and throughout chick embryogenesis.

Overexpression of v-ski or c-ski cDNAs has a pronounced effect on proliferation, morphological transformation and myogenic differentiation in cells in culture and in transgenic animals. Yet, little is known about expression of the c-ski locus or the relationship between c-ski cDNAs and alternatively spliced c-ski transcripts in chicken tissues, particularly in skeletal muscle or during embryogenesis. We developed a series of probes and oligonucleotide primers specific for the eight coding exons and the long 3' noncoding region found in chicken c-ski mRNAs. The most abundant chicken c-ski mRNAs in a vast array of tissues are 8.5 kb, with additional, but less abundant, mRNAs of 7.5, 6.5 and 4.4 kb. Steady-state levels of c-ski mRNAs, indistinguishable from transcripts in other tissues, accumulate in skeletal muscle from embryonic, newly hatched, and adult chicks. Only exon 2, a small exon of 111 bp, was found to be alternatively spliced in c-ski mRNAs. Transcripts with and without exon 2 appear in all tissues, in somites, and from the earliest stages of chick embryogenesis. Thus, c-ski cDNA sequences, which extend about 4.3 kb, represent either the least abundant form of c-ski mRNAs in tissues or a severely truncated form of the major 8.5 kb transcripts.

Animals↗

PubMed1995

Growth-restricted dengue virus mutants containing deletions in the 5' noncoding region of the RNA genome.

The dengue type 4 virus (DEN4) RNA genome contains a 101-nt 5' noncoding (NC) sequence which is predicted to form a stable secondary structure. DEN4 cDNA from which infectious RNA can be transcribed was used to engineer deletions in the 5' NC region for functional analysis of RNA structure and for isolation of DEN4 mutants that could be evaluated as candidates for use in a live attenuated vaccine. Eleven distinct deletions in the region of the DEN4 genome between nts 18 and 98 were constructed; each mutation was predicted to alter or disrupt the local base-parings in the 5' NC RNA structure. An infectious virus was not recovered from the RNA transcripts of five of these deletion mutants. Significantly, four of the five apparently lethal deletions were located in a 5- to 6-nt base-paired region of a predicted long stem or adjacent to it. In contrast, with one exception, mutants which yielded infectious virus had deletions which were located in a loop or short stem region. The effect of the deletions on the efficiency of translation of viral RNA transcripts was examined in vitro. The RNA transcripts of deletion constructs which did not yield viable virus were translated at an efficiency ranging from 40 to 160% that of wild-type virus transcripts. The translation efficiency of infectious RNA transcripts also varied. Deletion mutants recovered from RNA transcripts that exhibited low to moderate efficiency of translation had a small plaque morphology and exhibited reduced growth in simian LLC-MK2 and mosquito C6/36 cells compared to the wild-type virus. Among the 11 mutant constructs, deletion of nts 82-87 caused the greatest reduction in translation efficiency. Nevertheless, an infectious virus was recovered from LLC-MK2 cells transfected with the RNA transcripts of mutant d(82-87). The progeny of this mutant produced small plaques on LLC-MK2 cells and grew to low titer in these cells. Unlike wild-type DEN4 or other DEN4 deletion mutants tested, mutant d(82-87) failed to produce plaques on C6/36 cells and was also replication-defective in Aedes aegypti and Aedes albopictus following intrathoracic inoculation.

Aedes↗

PubMed1989

Histone genes in Physarum polycephalum: transcription and analysis of the flanking regions of the two H4 genes.

The histone H4 multigene family of Physarum polycephalum consists of two genes, H41 and H42. Both genes have an unusual structure in that they are interrupted by a small intron. The structure of the P. polycephalum H4 genes is discussed and compared to the structure of histone genes of other organisms. S1 nuclease analysis was used to map the 5' and 3' ends of the histone H4 messengers. We show that the histone H4 genes have a hybrid structure; they are interrupted by an intervening sequence, as in replacement variant histone genes of higher eukaryotes, but their 5' and 3' noncoding regions have the properties of replication-dependent histone genes: the 5' and 3' leader and trailer sequences are short, possess a 3'-hyphenated dyad symmetry element, and a CAGA sequence is found 3' to the hyphenated hairpin structure. This report also provides evidence that both genes are expressed in late G2 phase as well as in S phase and that their expression is temporally coordinated and quantitatively similar during the cell cycle.

Amino Acid Sequence↗

PubMed1992

Developmental expression of the Drosophila melanogaster calmodulin gene.

The highly conserved, intracellular calcium binding protein calmodulin is present in all cells at all times. In addition to this constitutive level, the amount of calmodulin is highly regulated according to the tissue or stage of development. Since there are only a few genes or a single gene for this protein in most species, intricate regulatory elements may be necessary to effect its complex regulation. This report adds new information concerning the gene structure and outlines the developmental and spatial regulation of Drosophila melanogaster calmodulin transcripts. The gene contains five exons, including a 49 bp exon in the 5' untranslated region, and spans over 16 kb. Homologues to this small, 5' noncoding exon have not been found in other calmodulin genes. The combined level of the transcripts is developmentally regulated, and the relative amounts of the two transcript size classes (1.65 kb and 1.9 kb) are differentially regulated during development. Primer extension experiments and RNase protection mapping show that both size classes of Drosophila calmodulin transcripts initiate at the same site but undergo alternative termination within the final exon. The spatial distribution of calmodulin transcripts was examined by in situ hybridization to sections of adults and to developmentally staged whole mount embryos. Calmodulin transcripts are evenly distributed early in embryogenesis. In later stages of embryogenesis, higher levels accumulate in the developing nerve cord and other tissues. Elevated levels of calmodulin transcripts are seen quite distinctly in the adult neural tissues and in the photoreceptor region of the compound eye.

Animals↗

PubMed1999

H4 acetylation, XIST RNA and replication timing are coincident and define x;autosome boundaries in two abnormal X chromosomes.

The inactive X (Xi) differs from its active homologue (Xa) in a number of ways, including increased methylation of CpG islands, replication late in S phase, underacetylation of histone H4 and association with XIST RNA. Global changes in DNA methylation occur relatively late in development, but the other properties all change during or shortly after the establishment of Xi and may play a role in the mechanism by which an inactive chromatin conformation spreads across most of the chromosome. In the present report, we use two human X;autosome translocation chromosomes to study the spreading of inactive X chromatin across X;autosome boundaries. In one of these chromosomes, t(X;6), Xp distal to p11.2 is replaced by 6p21.1-6pter and, in the other, ins(X;16), a small fragment derived from 16p13 is inserted into the distal third of Xq. In lymphoid cells from patients carrying these translocations in an unbalanced form, Xi was shown by HUMARA assay to be derived exclusively [t(X:6)] or predominantly [ins (X;16)] from the derived X chromosome. We used a combination of immunolabelling and RNA/DNA fluorescence in situ hybridization to define the distribution of XIST RNA, deacetylated H4 and late-replicating DNA across the two derived X chromosomes in inactive form. Within the limits of the cytogenetic techniques employed, the results show complete coincidence of these three parameters, with all three being excluded from the autosomal component of the derived X chromosome.

Acetylation↗

PubMed1997

Cloning of two candidate tumor suppressor genes within a 10 kb region on chromosome 13q14, frequently deleted in chronic lymphocytic leukemia.

Previous studies have indicated the presence of a putative tumor suppressor gene on chromosome 13q14, commonly deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). We have previously defined a minimally deleted region of 130 kb centromeric to the marker D13S272, and constructed a PAC and cosmid contig encompassing this area. In the present study we have made a detailed restriction and transcriptional map of the region of interest. Using these tools we have screened a panel of 206 primary CLL clones and three cell lines. In five CLL cases we found limited deletions defining the region of interest to an area of no more than 10 kb. Two adjacent genes, termed Leu1 and Leu2 (leukemia-associated gene 1 and 2), were mapped to the minimally deleted region, with several patients showing deletion borders within these genes. The Leu1 and Leu2 genes show little homology to previously published genes at the nucleotide and expected translated amino acid sequence level. Mutational analysis of the Leu1 and 2 genes in 170 CLL samples revealed no small intragenic mutations or point mutations. However, in all cases of 13q14 loss examined, the first exon of both genes, which are only 300 bp apart, were deleted. We conclude that the Leu1 and Leu2 genes are strong candidates as tumor suppressor gene(s) involved in B-CLL leukemogenesis.

Amino Acid Sequence↗

PubMed1991

Tissue-specific expression of multiple gamma-glutamyl transpeptidase mRNAs in rat epithelia.

gamma-Glutamyl transpeptidase (GGT) is an enzyme that plays a key role in interorgan glutathione transport. Three mRNAs (mRNAI, mRNAII, and mRNAIII) are known to encode the GGT precursor; they are initiated on three separate promoters on the single GGT gene. In this work, we identified by Northern blot and RNase H analysis a new GGT mRNA (mRNAIV). This mRNA differs from the others in its 5'-noncoding sequence. This mRNA species is the predominant GGT mRNA expressed in HTC hepatoma cells and in the small intestine in which its level increases from the base to the apex of the microvillus. The analysis of the GGT gene expression pattern in kidney, mammary gland, small intestine, liver, preneoplastic liver, and HTC hepatoma cells reveals a strong tissue or cell specificity. The mRNAIII was found in all the tissues and cells; in contrast, the expression of mRNAI, mRNAII, and mRNAIV is limited in normal tissues to the kidney and to the small intestine, the two tissues that display the highest enzyme activity. The synthesis of these three mRNAs is linked to the development of the kidney proximal tubule and to the differentiation of the enterocyte. The tissue and cell specificity of the GGT gene expression is based upon the use of multiple promoters that are controlled independently by specific cell factors.

Animals↗

PubMed1983

Cloning the heavy chain of human HLA-DR antigen using synthetic oligodeoxyribonucleotides as hybridization probes.

The recent development of the amino acid microsequence technique allows us to obtain partial sequence information using an extremely small amount of protein. Two sets of mixed oligonucleotide probes were chemically synthesized using the amino acid sequence information for the heavy chain of human HLA-DR antigen obtained by the microsequence technique. These two hybridization probes were used to screen cDNA clones constructed from cytoplasmic poly(A)+ mRNA from a human B lymphoblastoid homozygous cell line (LG-2). Of the 10,000 clones screened, two clones hybridized with the probes. DNA sequence analysis showed that the longer one of the two cDNA clones was 1183 nucleotides long, including the entire coding region, the signal peptide region, and the complete 3'-noncoding region. The deduced amino acid sequence of the HLA-DR alpha chain is identical to that of other cell lines with a different HLA-DR typing. However, several nucleotide differences are found in the 3'-untranslated region compared with that of other DR haplotypes.

Amino Acid Sequence↗

PubMed1997

An alfalfa rubisco small subunit homologue shares cis-acting elements with the regulatory sequences of the RbcS-3A gene from pea.

A genomic clone of RbcS was isolated from an alfalfa (Medicago sativa L. cv. Apica) genomic library and characterized. Although this clone has structural features similar to a functional gene, the second exon is interrupted by a stop codon and thus is not fully translatable in the plant. Sequence analysis of the 5' and 3' noncoding regions of RbcSK-1A showed a high sequence homology to the flanking sequences of the RbcS-3A gene from pea. The regions of homology contain many important cis-regulatory elements shown to be essential for regulation of the RbcS-3A gene in pea. The promoter of this alfalfa rubisco clone was used in a translational fusion to test its ability to control the expression of the GUS reporter gene in an homologous nuclear background. High levels of GUS enzyme activity were recorded. These strong levels are comparable to some exceptionally high levels produced in other studies following the use of photosynthesis gene promoters in fusions with the GUS reporter gene.

Base Sequence↗

PubMed1988

Multiple species of myeloperoxidase messenger RNAs produced by alternative splicing and differential polyadenylation.

Three clones of full-length cDNA encoding human myeloperoxidase were isolated from a human leukemia HL-60 cell cDNA library in lambda gt10 and characterized. Analysis of the nucleotide sequence of one of the cDNA clones, lambda MP-H17, indicated that the cDNA contained 3207 bp with an open reading frame of 2238 bp, a 5' noncoding region of 159 bp, a 3' noncoding region of 800 bp, and a poly(A) tail of 10 bp. cDNA of the two other clones, lambda MP-H7 and lambda MP-H14, each contained insertions with shorter sequences of 96 and 82 bp, respectively, on the open reading frame of lambda MP-H17 cDNA. A myeloperoxidase genomic clone was isolated, and the structure of its 5' region was determined and compared with the structures of these cDNAs. The comparison revealed that the three cDNAs were derived from myeloperoxidase mRNAs produced by alternative splicing from a transcript of the single gene. Nucleotide sequence analysis of the 3' region of the cDNAs of several clones indicated that the mRNAs were polyadenylated at five different sites. Amino acid sequence determination of the amino-terminal and carboxy-terminal portions of the myeloperoxidase light and heavy chains revealed that, during processing of a precursor polypeptide into the mature protein, the amino-terminal polypeptide, the small peptide between the light and heavy chains, and the carboxy-terminal amino acid were excised.

Amino Acid Sequence↗

PubMed1992

Alternate use of divergent forms of an ancient exon in the fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster.

The fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster contains three divergent copies of an evolutionarily conserved 3' exon. Two mRNAs encoding aldolase contain three exons and differ only in the poly(A) site. The first exon is small and noncoding. The second encodes the first 332 amino acids, which form the catalytic domain, and is homologous to exons 2 through 8 of vertebrates. The third exon encodes the last 29 amino acids, thought to control substrate specificity, and is homologous to vertebrate exon 9. A third mRNA substitutes a different 3' exon (4a) for exon 3 and encodes a protein very similar to aldolase. A fourth mRNA begins at a different promoter and shares the second exon with the aldolase messages. However, two exons, 3a and 4a, together substitute for exon 3. Like exon 4a, exon 3a is homologous to terminal aldolase exons. The exon 3a-4a junction is such that exon 4a would be translated in a frame different from that which would produce a protein with similarity to aldolase. The putative proteins encoded by the third and fourth mRNAs are likely to be aldolases with altered substrate specificities, illustrating alternate use of duplicated and diverged exons as an evolutionary mechanism for adaptation of enzymatic activities.

Amino Acid Sequence↗

PubMed1997

Disrupted IGF2 promoter control by silencing of promoter P1 in human hepatocellular carcinoma.

Previous investigations have supported or indicated a stimulatory role of the insulin-like growth factor II gene (IGF2) in hepatocarcinogenesis. We have studied the transcript levels, promoter usage, and imprinting status of the ICF2 gene and its relationship to H19 in human hepatocellular carcinomas (HCCs) and liver tumor cell lines. The activity of the IGF2 promoter P1 was lost in about 70% of the cases (18 of 25). This is the most prominent abnormality regarding the IGF2 regulation in this study. Total IGF2 as well as promoter P3 transcription were up-regulated in a small group of the tumors. Twenty genetically informative cases were obtained from 26 cases, thus excluding the probability of loss of heterozygosity of the IGF2 gene. Among these, nine showed abnormal monoallelic expression of IGF2. One HCC and one HCC cell line proved loss of functional imprinting of IGF2. H19 and IGF2 were regulated in parallel, and expression levels were variable. Taken together, the disruption of the IGF2 promoter regulation, particularly the loss of P1 activity, is a common feature of human HCCs. The loss of P1 activity explains the frequent loss of biallelic IGF2 expression and may potentially be used as a diagnostic or monitoring marker for human HCC.

Alleles↗

PubMed1998

Gene and cDNA structures of flounder insulin-like growth factor-I (IGF-I): multiple mRNA species encode a single short mature IGF-I.

To understand the comprehensive mechanisms of gene expression and processing for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the gene organization, promoter and transcriptional initiation sites, alternative splicing and polyadenylating sites, and the cDNA structures of this gene in the Japanese flounder, Paralichthys olivaceus. The flounder IGF-I gene was found to be composed of five exons and four introns spanning 17.5 kb. By Northern blot analysis, two major mRNA classes of 4.7 kb and 2.9 kb were found in the liver. cDNA cloning and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that these two mRNA classes result from two different-sized 3'-noncoding regions generated by alternative usage of two polyadenylating signals. Further analysis by RT-PCR and sequencing revealed that these mRNA classes both contain two subclasses of mRNA encoding two forms of IGF-I prepropeptide, preproIGF-I-1 and preproIGF-I-2. The two forms of preproIGF-I share the identical signal peptide and mature IGF-I domain but contain different E domains as a result of alternative splicing in exon 3. The mature form of flounder IGF-I was found to comprise 68 amino acid residues, showing a small molecular weight, 7486. In the 5'-flanking region, one major and four minor transcription start sites have been identified by ribonuclease protection assay between -230 and -130 from the translation initiation codon, but no canonical TATA box or GC box was detected in their upstream regions up to -724. The results suggest that some unknown transcription initiation factors are functioning in the promotion of IGF-I gene expression.

Amino Acid Sequence↗

PubMed1995

The chicken beta A4- and beta B1-crystallin-encoding genes are tightly linked.

Analysis of the 5' flanking region of the chicken beta B1-crystallin-encoding gene (beta B1-cry) revealed regions of sequence homology with the bovine beta A4-crystallin-encoding gene (beta A4-cry). Subsequently, the chicken beta A4-cry cDNA sequence was determined, and it was demonstrated that beta A4- and beta B1-cry are linked head to head in the chicken chromosome with 2147 nucleotides (nt) of intergenic spacer. Chicken beta A4-cry contains six exons, with the first exon being noncoding. Chicken beta A4-cry is the smallest beta-cry ever described, due to the small size of its introns which range in length from 68 to 96 nt. While three polymorphisms were noted between some cDNA clones and the genomic sequence, Southern blot analysis demonstrated that beta A4-cry exists as a single copy in the chicken genome. Northern blot analysis indicated that beta A4-cry is a lens-specific transcript which is expressed at higher levels in the embryo than in the adult. The beta A4-cry mRNA is present at 400-fold lower levels than the beta B1-cry mRNA in the 14-day embryonic chicken lens, and at 2000-fold lower levels than the beta B1-cry mRNA in the adult lens. These results are consistent with the idea that the beta-cry family was once clustered in the chromosome as the gamma-cry family is today, and raises the possibility that the relatively low expression of beta A4-cry is mechanistically linked to the high expression of beta B1-cry in the chicken lens.

Animals↗

PubMed1991

Nucleotide sequence and evolution of coding and noncoding regions of a quail mitochondrial genome.

Segments of the Japanese quail mitochondrial genome encompassing many tRNA and protein genes, the small and part of the large rRNA genes, and the control region have been cloned and sequenced. Analysis of the relative position of these genes confirmed that the tRNA(Glu) and ND6 genes in galliform mitochondrial DNA are located immediately adjacent to the control region of the molecule instead of between the cytochrome b and ND5 genes as in other vertebrates. Japanese quail and chicken display another distinctive characteristic, that is, they both lack an equivalent to the light-strand replication origin found between the tRNA(Cys) and tRNA(Asn) genes in all vertebrate mitochondrial genomes sequenced thus far. Comparison of the protein-encoding genes revealed that a great proportion of the substitutions are silent and involve mainly transitions. This bias toward transitions also occurs in the tRNA and rRNA genes but is not observed in the control region where transversions account for many of the substitutions. Sequence alignment indicated that the two avian control regions evolve mainly through base substitutions but are also characterized by the occurrence of a 57-bp deletion/addition event at their 5' end. The overall sequence divergence between the two gallinaceous birds suggests that avian mitochondrial genomes evolve at a similar rate to other vertebrate mitochondrial DNAs.

Amino Acid Sequence↗

PubMed1994

Major human epididymis-specific gene product, HE3, is the first representative of a novel gene family.

Differential screening of a human epididymal cDNA library led to the isolation and characterization of a major epididymis-specific cDNA clone family, referred to as HE3. More detailed sequence and PCR analysis identified two different but homologous gene transcripts, HE3 alpha and HE3 beta. The former represents an mRNA of ca. 1 kb, encoding a putative small secretory polypeptide of 14903 MW. The HE3 beta transcript was only found as incomplete 3' fragments. Analysis of human genomic DNA by Southern blotting suggested the presence in the human genome of at least three independent HE3-related genes. Isolation of genomic clones for the HE3 alpha gene showed this to contain a single intron of 1.4 kb in the 5' noncoding region. Although genomic clones corresponding to HE3 beta could not be found, a third highly homologous gene, HE3 gamma, was identified as a potential pseudogene. Neither nucleotide nor encoded amino acid sequences of the HE3 gene family are related to any other known sequence in the central databases, and thus represents a novel human gene family, with at least three nonallelic members. Northern hybridization analysis showed that HE3 gene products are specifically expressed in the human epididymis, and not in any other tissue examined. Furthermore, except for the pig, no other nonprimate species has been identified to express homologous sequences in the epididymis. RNase protection assays showed that both the HE3 alpha and HE3 beta, but not the HE3 gamma genes, are expressed in the human epididymis.

Amino Acid Sequence↗

PubMed1992

Characterization of the rabbit CYP1A1 and CYP1A2 genes: developmental and dioxin-inducible expression of rabbit liver P4501A1 and P4501A2.

In adult rabbits, the CYP1A1 and CYP1A2 genes are expressed constitutively. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to elevations in both CYP1A1 and CYP1A2 gene products (S. T. Okino et al., 1985, Proc. Natl. Acad. Sci. USA 82, 5310-5314). In this report, we have characterized the rabbit CYP1A1 and CYP1A2 genes, and analyzed the pattern of expression of these genes in neonatal animals following exposure to TCDD. Genomic clones encoding the entire rabbit CYP1A1 and CYP1A2 genes were characterized. Restriction enzyme analysis and partial DNA sequence analysis identified the seven exons for the CYP1A1 and CYP1A2 genes. Primer extension analysis using mRNA from TCDD-treated neonatal rabbits helped confirm the start of transcription for the CYP1A genes. The length of the noncoding first exon of the CYP1A1 gene was 74 bases, compared to 90 and 88 bases for the human and rodent CYP1A1 genes. The length of the noncoding CYP1A2 gene first exon was 53 bases, similar to its counterpart in human and rodents. DNA sequence analysis of the 5' regulatory regions and comparison to the rodent and human CYP1 genes demonstrated that the rabbit CYP1A1 and CYP1A2 genes were most similar to their human orthologs. The 5' region of the CYP1A1 gene contained several consensus dioxin (Ah)-receptor responsive elements (XREs), while no functional XRE sequences were identified in the CYP1A2 gene. When expression of the two genes were monitored, a small amount of constitutive P4501A1 mRNA was detected in neonatal rabbits from the ages of 1 to 17 days, while P4501A2 mRNA levels could not be observed until 8-12 days postpartum. In response to TCDD treatment, P4501A1 mRNA levels were inducible at all neonatal time points, while P4501A2 mRNA levels could not be induced until the animals were 3-5 days postpartum. While the dioxin Ah-receptor most likely plays a major role in the induction of these genes by TCDD, early expression of the CYP1A1 and CYP1A2 genes is differentially regulated in a developmental fashion.

Aging↗