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PubMed1986

Human cytochrome P-450 4 mRNA and gene: part of a multigene family that contains Alu sequences in its mRNA.

Several overlapping lambda gt11 cDNA clones have been sequenced and shown to encode for the full-length human cytochrome P-450 4. The structure and location of the exons and flanking intron regions were also identified from a lambda EMBL-3 human genomic clone that encodes the full-length human P-450 4 gene. The human P-450 4 mRNA is flanked by 62 base pairs of 5'- and 1508 base pairs of 3'-noncoding sequence, with 1548 bases that encode a protein of 516 amino acids (Mr, 58,376). The predicted amino acid sequence of human P-450 4 is 69% and 70% homologous to its equivalent in mouse and rat, respectively, 75% homologous to rabbit P-450 4, and 68% homologous to human P1-450. The 7.6-kilobase gene encodes 3118 nucleotides of exon sequence that is separated by six introns into seven exons. Exon 7, which is 1802 nucleotides, contains three inverse/complement Alu sequences that are organized in tandem. Comparison of the genomic DNA sequence of the human P-450 4 gene with the human P1-450 and related genes in rat and mouse and the identification of the amino acid residues and triplet codon at each exon-intron junction show that the location of each intron in the human P-450 4 gene is conserved within this gene family. Although the length and homology of the introns within a related gene family may not be conserved, the location of intronic sequences may be an important determinant in the identification of related P-450 genes.

Amino Acid Sequence↗
PubMed1987

The psbA gene of DCMU-resistant Euglena gracilis has an amino acid substitution at serine codon 265.

The psbA gene coding for the herbicide binding QB protein of photosystem II has been sequenced previously (Karabin et al. 1984). A herbicide resistant mutant of Euglena, Euglena gracilis ZR, was studied by sequencing part of the psbA gene and its corresponding mRNA. Sequencing reactions were done by annealing a psbA specific, end-labeled DNA-oligomer to total chloroplast DNA or RNA and extending this primer with reverse transcriptase in the presence of the four dideoxynucleotides. An amino acid substitution from serine to alanine at position 265 was detected. All known herbicide resistant higher plants sequenced to date and the Chlamydomonas mutant DCMU-4 show a change at exactly this same position, but the substitution in higher plants is from serine to glycine.

Amino Acid Sequence↗
PubMed1985

Characterization of the messenger RNA population coding for components of viperid snake venom.

Total polyadenylated messenger RNA was prepared from the milked venom glands of the South African puff adder (Bitis arietans) and translated in an in vitro translation system. The products of cell free synthesis were immunoprecipitable with puff adder venom antiserum. Treatment of these cell free products with a dog pancreas microsomal membrane preparation demonstrated the presence of signal peptides. Northern blot hybridization of total puff adder venom gland mRNA to its complementary single stranded copy DNA revealed two discrete mRNA populations coding for the major components of puff adder venom. The relative amounts of these polyadenylated mRNA sequences changed during the onset of venom synthesis, suggesting mRNA deadenylation, general endonucleolytic RNA degradation and selective degradation of high molecular weight message components.

Animals↗
PubMed1983

The accumulation of delta-crystallin mRNA in transdetermination and transdifferentiation of neural retina cells into lens.

By means of hybridization with DNA complementary to delta-crystallin mRNA (delta-mRNA) sequences (delta-cDNA), the levels of delta-mRNA in three different culture systems of 8-day-old chick embryonic neural retina were determined. After 30 days of culturing in vitro, the level of delta-mRNA in cells cultured under conditions of spreading cultures (SpC) was 50 times higher than in the cells maintained in aggregate cultures (AgC) throughout. When the cells pre-cultivated in SpC for an initial 10 days were transferred into AgC, the delta-mRNA level in 30-day cultures was 40 times higher than that in 3-day SpC. The level of delta-mRNA in neural retina in situ was negligible, but it became detectable in 10-day SpC. The initial appearance of detectable delta-mRNA in 10-day SpC coincides with the timing of 'transdetermination' of neural retina cells into lens cells.

Animals↗
PubMed1998

The expression of Huntingtin-associated protein (HAP1) mRNA in developing, adult and ageing rat CNS: implications for Huntington's disease neuropathology.

Using radioactive in situ hybridization, we have mapped the expression of Huntingtin-associated protein (HAP1) mRNA in rat brain at developmental stages (E12-E19, PO-P21), in adult rats (3 months) and in 'aged' (19-21 months) rats. Using two pairs of 45mer oligonucleotide probes specific for HAP1A and a probe which recognizes regions of both the HAP1A and HAP1B mRNA sequences (panHAP1), we find that the expression of HAP1 mRNA is specific to the CNS and restricted predominantly to anatomically connected limbic structures, particularly the amygdala (medial and corticomedial nuclei), the hypothalamus (arcuate, preoptic, paraventricular and lateral hypothalamic area), bed nucleus of the stria terminalis (BNST) and the lateral septal nuclei. HAP1 mRNA was detected in embryos at E12 and displayed a prevalent distribution in the developing limbic structures by E15. In aged, 19-21-months-old, rats there is a downregulation of HAP1 mRNA expression across all CNS loci where HAP1 was previously abundant. The lowest levels of HAP1 mRNA expression corresponded with the areas of greatest pathological cell loss in Huntington's disease (HD); the caudate putamen, globus pallidus and neocortex. These observations support the suggestion that HAP1 plays an important role in the neuropathology of HD.

Aging↗
PubMed1976

A general method for cloning eukaryotic structural gene sequences.

Complementary DNA, transcribed in vitro from purified rabbit globin messenger RNA and made double-stranded, has been inserted into Escherichia coli plasmids pSC101 and pMB9 by the poly(dT)/poly(dA) "tailing" and annealing technique. E. coli transformants given by this DNA preparation have been shown to contain globin sequences by the hybridization of globin RNA to DNA from clones grown and lysed in situ on nitrocellulose filters. An estimate of the amount of inserted globin sequences has been provided by fingerprint analysis of globin mRNA sequences hybridized to the purified plasmid chimeras. Inserted sequences so far subjected to detailed analysis have been ascribed to the rabbit beta globin chain. The susceptibility of inserted beta globin, sequences to the restriction endonuclease EcoRI confirms the existence of a site already found through previous nucleotide sequence analysis.

Base Sequence↗
PubMed1984

Isolation of cloned cDNA encoding mammalian ornithine decarboxylase.

During the growth of mammalian cells the level of ornithine decarboxylase ( OrnDCase ; L-ornithine carboxy-lyase, EC 4.1.1.17), the first enzyme in polyamine biosynthesis, undergoes rapid changes. As an initial step in the study of possible genetic mechanisms involved in these changes, we have isolated cDNA clones encoding OrnDCase . To obtain RNA enriched for OrnDCase messenger, mouse myeloma cells that overproduce OrnDCase were selected in the presence of the OrnDCase inhibitor, difluoromethylornithine. A pBR322 cDNA library was prepared from poly(A)+ RNA isolated from difluoromethylornithine-resistant cells, and the library was probed with [32P]cDNA representing mRNA sequences from resistant or parental (sensitive) cells. All clones hybridizing preferentially to the resistant cell probe shared nucleotide sequences. A representative clone containing 1.1 kilobases of cDNA was shown to encode OrnDCase sequences by in vitro translation of hybrid-selected mRNA followed by precipitation of the translation products with anti- OrnDCase antiserum. Using this cDNA clone as a probe, we found that mouse DNA yielded several restriction fragments that react with the OrnDCase cDNA. In the difluoromethylornithine-resistant myeloma cells, one of these DNA segments is amplified and the level of OrnDCase mRNA is greatly increased compared with that in parental plasmacytoma cells. The level of OrnDCase mRNA is also increased in cultured 3T3 cells stimulated with serum and in mouse kidneys after administration of androgen, indicating that OrnDCase gene transcription and/or mRNA stability are regulated during cell growth.

Animals↗
PubMed2003

Analysis of 5'-end sequences of chimpanzee cDNAs.

We constructed full-length enriched cDNA libraries from chimpanzee brain, skin, and liver tissues by the oligo-capping method to establish a database of sequences of chimpanzee genes. Randomly selected clones from the libraries were subjected to one-pass sequencing from their 5'-ends. As a result, we collected 6813 chimpanzee cDNA sequences longer than 400 bp. Homology search against human mRNA sequences (RefSeq mRNAs) revealed that our collection included sequences of 1652 putative chimpanzee genes. In order to calculate the sequence identity between human and chimpanzee homologs, we constructed 5'-end consensus sequences of 226 chimpanzee genes by aligning at least three sequences for individual genes. Sequence identity was estimated by comparing these consensus sequences and the corresponding sequences of their human homologs. The average sequence identity of the 5'-end cDNAs was 99.30%. Those of the 5'-UTRs and CDSs were 98.79% and 99.42%, respectively. The results confirmed that human and chimpanzee genes are highly conserved at the nucleotide level. As for amino acids, the average sequence identity was 99.44%. The average synonymous (K(S)) and nonsynonymous (K(A)) divergences were estimated to be 1.33% and 0.28%, respectively.

5' Flanking Region↗
PubMed2003

Growth hormone regulation of glomerular AT1 angiotensin receptors in adult uninephrectomized male rats.

Sex differences exist in the mechanisms initiating early compensatory renal growth after unilateral nephrectomy (UNX); remnant kidney growth is growth hormone (GH) independent in adult female rats and GH dependent in adult male rats. The present study determined whether sex differences also exist in angiotensin type 1 receptor (AT1R) regulation during early remnant kidney (REM) growth after UNX, and if so, whether GH modulates AT1R expression after UNX in the male rat. Scatchard analysis of radioligand binding in glomeruli demonstrated that 48 h post-UNX, AT1R density (Bmax) was significantly decreased by 20% in female REM compared with control kidneys. In contrast, male REM glomerular Bmax was significantly increased by 28% compared with control kidneys. Furthermore, GH-suppressed male rats displayed attenuated REM growth, which was associated with a 35% decrease in AT1R Bmax. Losartan treatment also decreased REM AT1R Bmax by 55%. The activity of mRNA binding proteins that bind to the 5' leader sequence of the AT1R was regulated by UNX and GH treatment in an inverse manner to AT1R expression. These findings suggest that in rats 1) there are sex differences in the regulation of glomerular AT1R expression after UNX; 2) the increase in AT1R binding sites in the male REM is regulated by GH and mediates early remnant kidney growth; and 3) AT1R 5' leader sequence mRNA binding proteins play a role in UNX and GH regulation of glomerular AT1Rs in both males and females.

Adaptation, Physiological↗
PubMed1982

Induction of the messenger ribonucleic acid coding for phosphoenolpyruvate carboxykinase in H4-II-E cells. Evidence for a nuclear effect of cyclic AMP.

The effect of N6,O2'-dibutyryl cyclic adenosine monophosphate (Bt2cAMP) on the induction of the mRNA coding for the enzyme phosphoenolpyruvate carboxykinase was examined in H4-II-E cells. this mRNA comprised about 0.1% of total cellular poly(A)+RNA activity in uninduced cells and was increased 5- to 7-fold by the cyclic nucleotide. The maximal level was reached 3 h after addition of the nucleotide to the cell culture. This induction is attributed to cAMP since the nonmetabolizable analogs 8-bromocAMP and 8-(4-chlorophenylthio)cAMP produce inductions comparable to Bt2cAMP while sodium butyrate and dibutyryl cyclic GMP had little effect. The increased translational activity correlated well with a proportionate increase in the amount of phosphoenolpyruvate carboxykinase (P-enolpyruvate carboxykinase) mRNA sequences which were hybridizable to a specific cDNA probe. Blot hybridization of total nuclear RNA isolated from uninduced H4-II-E cells revealed eight P-enolpyruvate carboxykinase RNA sequence species ranging in size from 1.8 to 6.9 kilobases. Treatment with Bt2cAMP increased the amount of all eight of these forms. This increase became maximal by 45-60 min and was maintained for at least 1 h. In contrast, analysis of cytoplasmic RNA showed a single 3.2-kilobase (23 S) band, which was still increasing in amount 2 h after Bt2cAMP treatment. Thus, Bt2cAMP resulted in a sequential induction of nuclear P-enolpyruvate carboxykinase RNA sequences followed by an increase in cytoplasmic phosphoenolpyruvate carboxykinase mRNA. We conclude that cyclic AMP exerts its main effect on P-enolpyruvate carboxykinase induction at the nuclear level.

Animals↗
PubMed1994

Structure and expression of mRNA for vitellogenin in Bombyx mori.

Vitellogenin, a precursor of major yolk protein of the silkworm, Bombyx mori is a tetramer composed of each two molecules of heavy and light subunits. We cloned mRNA sequence for the B. mori vitellogenin and analyzed its structure. Sequence alignment of several overlapping cDNA clones indicated that the vitellogenin mRNA is approx. 5.7 kb, containing an open reading frame for a peptide with 1782 amino acid residues. By comparing the deduced amino acid sequence with the amino-terminal primary structures of vitellogenin subunits, it is suggested that the heavy and light subunits of the B. mori vitellogenin are encoded by a single contiguous mRNA. The primary translation product of the vitellogenin mRNA was detected in the microsomal fraction prepared from the fat body of vitellogenic females. Northern blot analysis of the fat body RNA demonstrated that the biosynthesis of vitellogenin in B. mori is regulated in a tissue-, sex- and stage-specific manner at the level of mRNA. Possible cause for discrepancy between the present results and our previous proposal (Izumi, S. and Tomino, S. (1983) Insect Biochem. 13, 81-85) on the biosynthesis of B. mori vitellogenin is also discussed.

Amino Acid Sequence↗
PubMed1987

Oestrogen regulates cathepsin D mRNA levels in oestrogen responsive human breast cancer cells.

A cDNA library was constructed from the mRNA of the ZR-75 oestrogen responsive human breast cancer cell line and screened for oestrogen regulated mRNA sequences. Of the recombinants isolated, 30 contained cDNA that corresponded to a single mRNA species of 2.1 kb that was induced between 10 and 15 fold by oestradiol treatment. The sequence of the entire open reading frame and 3' non-coding region of the mRNA was determined and shown to encode the aspartyl protease cathepsin D. The induction of cathepsin D mRNA is specific for oestrogen and is maximal at 3 X 10(-10) M. Cathepsin D mRNA levels were increased by oestrogen in 3 oestrogen responsive breast cancer cell lines. Cathepsin D mRNA was expressed but not regulated in an oestrogen receptor negative breast tumour cell line, BT 20, and in 2 other malignant cell lines, Hela and A431.

Base Sequence↗
PubMed1988

Expression of specific isoforms of protein 4.1 in erythroid and non-erythroid tissues.

Protein 4.1 in red cells is an important submembrane linking protein that binds to spectrin actin complexes at one end of its structure and to transmembrane proteins, such as glycophorin, at the other. Protein 4.1 thus contributes to the strength and flexibility of the erythrocyte membrane, a fact dramatically exemplified by the appearance of hereditary hemolytic anemias in patients with absent or abnormal protein 4.1. Recently, protein 4.1 forms have been discovered in many non-erythroid tissues. Their intracellular locations raise the possibility that these isoforms might have different functions. We have thus conducted comparative analysis of erythroid and non-erythroid protein 4.1 forms by cloning and sequencing erythroid and lymphoid protein 4.1 cDNAs. The lymphoid protein 4.1 isoforms exhibit at least five nucleotide sequence motifs that appear to be either inserted or deleted relative to the erythroid mRNA sequence by alternative splicing of a common mRNA precursor. One of these motifs, located within the spectrin-actin binding domain, is found only in erythroid cells and is specifically produced during erythroid cell maturation. The selective expression of this alternatively spliced mRNA during erythroid maturation implies the existence of a lineage specific splicing mechanism whose activity is triggered by terminal maturation. Two motifs alter the 5' untranslated region of the "prototypical" erythroid mRNA in such a way as to permit synthesis of a novel larger isoform. This form appears to localize preferentially in the nucleus. We thus conclude that a single gene gives rise to multiple protein 4.1 isoforms with potentially diverse locations and functions.

Base Sequence↗
PubMed2004

Developing RNase P ribozymes for gene-targeting and antiviral therapy.

RNase P, a tRNA processing enzyme, contains both RNA and protein subunits. M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, recognizes its target RNA substrate mainly on the basis of its structure and cleaves a double stranded RNA helix at the 5' end that resembles the acceptor stem and T-stem structure of its natural tRNA substrate. Accordingly, a guide sequence (GS) can be covalently attached to the M1 RNA to generate a sequence specific ribozyme, M1GS RNA. M1GS ribozyme can target any mRNA sequence of choice that is complementary to its guide sequence. Recent studies have shown that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1 and human cytomegalovirus, and the BCR-ABL oncogenic mRNA in vitro and effectively reduce the expression of these mRNAs in cultured cells. Moreover, an in vitro selection scheme has been developed to select for M1 GS ribozyme variants with more efficient catalytic activity in cleaving mRNAs. When expressed in cultured cells, these selected ribozymes also show an enhance ability to inhibit viral gene expression and growth. These recent results demonstrate the feasibility of developing the M1GS ribozyme-based technology as a promising gene targeting approach for basic research and clinical therapeutic application.

Antiviral Agents↗
PubMed1986

Amino acid sequence of the testosterone-regulated mouse kidney RP2 protein deduced from its complementary DNA sequence.

The major forms of testosterone-regulated RP2 messenger RNA (also known as MAK mRNA and pMK908) in the mouse kidney were characterized by examining cDNA and genomic clones. Three sizes of RP2 mRNA are detected by Northern blot analysis and these were shown to result from polyadenylation at three distinct sites within the primary transcript of this single-copy gene. The complete RP2 mRNA sequence was obtained from overlapping cDNA clones, revealing an open reading frame of 357 amino acids that corresponds to a protein of 40,365 daltons. The detection of RP2 mRNA in all tissues examined to date suggests that the RP2 protein may function in a housekeeping role in all cells. This is supported by the finding of a high percentage of G + C residues at the 5' end of the gene, including a sequence homologous to the binding site of the transcription factor Sp1, which has been suggested to affect the regulation of other housekeeping genes that have been characterized. An examination of the amino acid sequence indicates that the RP2 protein is proline-rich and is composed of alternating alpha-helix and beta-sheet regions. RP2 is probably not integrated into a membrane structure in the cell as it does not appear to contain hydrophobic regions capable of spanning a membrane.

Amino Acid Sequence↗
PubMed2005

Identification and characterization of RNA sequences to which human PUMILIO-2 (PUM2) and deleted in Azoospermia-like (DAZL) bind.

Members of the Pumilio and DAZL family of RNA binding proteins are required for germ cell development in Drosophila, Xenopus, and Caenorhabditis elegans. Here, we report identification and characterization of RNA sequences to which PUM2 and DAZL bind. We established that human PUM2 specifically recognized the Drosophila Pumilio RNA target (the NRE or Nanos regulator element sequence); single nucleotide changes in the NRE abolished PUM2 binding. Then, we used coimmunoprecipitation to isolate human transcripts specifically bound by PUM2 and DAZL and subsequently identified those that contain NRE-like sequence elements. We confirmed that the interacting proteins, PUM2 and DAZL, are capable of binding the same RNA target and further characterized mRNA sequences bound by both proteins in the 3'UTR of human SDAD1 mRNA. Taken together, the results define sequences to which these germ cell-specific RNA binding proteins may bind to promote germ cell development.

Animals↗
PubMed1983

DNA sequence organization and transcription of the chicken genome.

A new approach has been used to examine DNA sequence organization in the chicken genome. The interspersion pattern was determined by studying the fraction of labelled DNA fragments of different lengths that hybridized to an excess of short chicken repeated DNA sequences. The results indicate that chicken DNA has a pattern of sequence organization quite different than the standard 'Xenopus' or 'Drosophila' patterns. Two classes of unique sequences are found. One, 34% of the genome, consists of unique sequences approx. 4 kb long interspersed with repeated sequences. The second, non-interspersed fraction, 38% of the genome, consists of unique sequences found in long tracts, a minimum of approx. 22 kb in length. In an attempt to determine whether a relationship exists between DNA sequence organization and the distribution of structural genes we have isolated chicken DNA sequences belonging to different interspersion classes and tested each for the presence of structural genes by hybridization to excess poly(A)+ mRNA. Sequences complementary to poly(A)+ mRNA can be found with approximately the same frequency in both the non-interspersed fraction of the genome and a repeat-contiguous fraction enriched for interspersed sequences.

Animals↗
PubMed1980

The messenger RNA sequences in human fibroblast cells induced with poly rI.rC to produce interferon.

Induction of human fibroblast cells with poly rI.rC induces interferon mRNA which can be translated into interferon precursor in wheat germ cell free system or in Xenopus oocytes into biologically active interferon. The extent of gene expression in the poly rI.rC induced cells was compared to that of the uninduced cells by hybridization of the mRNA to complementary DNA. Homologous template driven hybridization of cDNA revealed the presence of two clearly defined transitions in the total poly A RNA from the induced cells; abundant class and a scarce class comprising approximately 37,000 diverse species of RNA. Heterologus hybridization of the cDNA with total uninduced mRNA showed that the majority of the mRNA sequences are the same in both the induced and uninduced cells. The results of the hybridization using cDNA prepared to the fraction enriched for interferon mRNA, however, showed that about 4% of the sequences present in the interferon enriched fraction are not present in the uninduced cells. These differences may result from the poly rI.rC induced alterations in gene expression.

Fibroblasts↗