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PubMed1990

De novo synthesis and secretion of prolactin-like protein-B by rat placental explants.

Mid- to late-gestation rat placenta expresses three PRL-related mRNAs, rat placental lactogen-II (rPL-II), rat PRL-like protein-A (rPLP-A), and rat PRL-like protein-B (rPLP-B). The protein product of rPL-II mRNA has been characterized, and the protein products of the rPLP-A mRNA were recently identified. The mol wt of a nonsecreted nonglycosylated rPLP-B protein would be 27,145 based on the mRNA sequence. The present study is the first to report the identification of the rPLP-B protein. Antiserum was generated against a chemically synthesized oligopeptide inferred from a specific region of the rPLP-B cDNA. Three or four distinct proteins synthesized and secreted by rat basal zone explants (day 15 gestation) showed cross-reactivity with the rPLP-B antiserum. The relative mol wt of these immunoreactive proteins is approximately 30,000, with a pI varying from 6.1-6.6. De novo synthesized rPLP-B proteins were not secreted by the explant tissue in the presence of tunicamycin, suggesting that the proteins are glycosylated. These data are consistent with the presence of one potential N-glycosylation site derived from the rPLP-B mRNA sequence. The rPLP-B antiserum showed no cross-reactivity with proteins identified using antisera against rPLP-A, rPL-II, or human pregnancy-specific beta 1-glycoprotein. Immunocytochemical studies were carried out using paraffin sections from placentas of day 14 and 17 pregnant rats which were treated with anti-rPLP-B, followed by avidin-biotin-peroxidase complex. These experiments show perinuclear staining, which was localized in basophilic cytotrophoblast cells, confirming previous in situ mRNA hybridization studies. Although no physiological role has been established for rPLP-B, the synthesis and secretion of this protein by cells in contact only with maternal circulation suggest a hormonal role.

Amino Acid Sequence↗
PubMed1989

Expression of two kallikrein gene family members in the rat prostate.

We have characterized two kallikrein gene family members expressed in the prostate and submaxillary glands of rats. One mRNA (S3) is identical with the previously characterized submaxillary gland S3 mRNA that encodes an enzyme closely related to tonin. The second mRNA (P1) encodes a novel kallikrein-like enzyme that retains key amino acid residues responsible for the characteristic enzymatic cleavage specificity of kallikrein. Two P1-specific oligonucleotide probes derived from the P1 mRNA sequence were used to demonstrate the presence of P1 mRNA in the prostate and submaxillary glands and its absence in eight other rat tissues known to express one or more members of the kallikrein family. The P1-coding gene (rGK-8) was identified among genomic clones containing kallikrein family members by hybridization with a P1-specific oligonucleotide probe. The identification of the P1 gene was verified by nucleotide sequencing; the exon sequences of rGK-8 match the P1 mRNA sequence. The upstream region of rGK-8, where transcriptional regulatory elements likely reside, is very similar to that of other rat kallikrein family genes which are expressed in distinct tissue-specific patterns.

Amino Acid Sequence↗
PubMed1983

Effects of adenovirus infection on rRNA synthesis and maturation in HeLa cells.

The production of cytoplasmic and nucleolar rRNA species was examined in HeLa cells infected with high multiplicities of adenovirus type 5. Both 28S and 18S rRNA newly synthesized in infected cells ceased to enter the cytoplasm as reported previously (N. Ledinko, Virology 49: 79-89, 1972; H. J. Raskas, D. C. Thomas, and M. Green, Virology 40: 893-902, 1970). However, the effects on 28S cytoplasmic rRNA were observed considerably earlier in the infectious cycle than those on 18S rRNA. The inhibition of cellular protein synthesis and of the appearance in the cytoplasm of labeled cellular mRNA sequences (G. A. Beltz and S. J. Flint, J. Mol. Biol. 131: 353-373, 1979) were also monitored in infected cultures. During the later periods of an infectious cycle, from 18 h after infection, nucleolar rRNA synthesis and processing and exit of 18S rRNA from the nucleus were inhibited, probably reflecting the failure of infected cells to synthesize normal quantities of ribosomal proteins. The earliest responses of cellular RNA metabolism to adenovirus infection were, however, the rapid and apparently coordinate reductions in the levels of newly synthesized 28S rRNA and cellular mRNA sequences entering the cytoplasm.

Adenoviridae Infections↗
PubMed2006

Evidence for conservation and selection of upstream open reading frames suggests probable encoding of bioactive peptides.

BACKGROUND: Approximately 40% of mammalian mRNA sequences contain AUG trinucleotides upstream of the main coding sequence, with a quarter of these AUGs demarcating open reading frames of 20 or more codons. In order to investigate whether these open reading frames may encode functional peptides, we have carried out a comparative genomic analysis of human and mouse mRNA 'untranslated regions' using sequences from the RefSeq mRNA sequence database. RESULTS: We have identified over 200 upstream open reading frames which are strongly conserved between the human and mouse genomes. Consensus sequences associated with efficient initiation of translation are overrepresented at the AUG trinucleotides of these upstream open reading frames, while comparative analysis of their DNA and putative peptide sequences shows evidence of purifying selection. CONCLUSION: The occurrence of a large number of conserved upstream open reading frames, in association with features consistent with protein translation, strongly suggests evolutionary maintenance of the coding sequence and indicates probable functional expression of the peptides encoded within these upstream open reading frames.

3' Untranslated Regions↗
PubMed2002

Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design.

Use of antisense oligonucleotides is a versatile strategy for achieving control of gene expression. Unfortunately, the interpretation of antisense-induced phenotypes is sometimes difficult, and chemical modifications that improve the potency and specificity of antisense action would be useful. The introduction of locked nucleic acid (LNA) bases into oligonucleotides confers exceptional improvement in binding affinity, up to 10 degrees C per substitution, making LNAs an exciting option for the optimization of antisense efficacy. Here we examine the rules governing antisense gene inhibition within cells by oligonucleotides that contain LNA bases. LNA- containing oligomers were transfected into cells using cationic lipid and accumulated in the nucleus. We tested antisense gene inhibition by LNAs and LNA-DNA chimeras complementary to the 5'-untranslated region, the region surrounding the start codon and the coding region of mRNA, and identified effective antisense agents targeted to each of these locations. Our data suggest that LNA bases can be used to develop antisense oligonucleotides and that their use is a versatile approach for efficiently inhibiting gene expression inside cells.

5' Untranslated Regions↗
PubMed1989

Molecular phenotypes in cultured maple syrup urine disease cells. Complete E1 alpha cDNA sequence and mRNA and subunit contents of the human branched chain alpha-keto acid dehydrogenase complex.

The activity of the branched-chain alpha-keto acid dehydrogenase complex is deficient in patients with the inherited maple syrup urine disease (MSUD). To elucidate the molecular basis of this metabolic disorder, we have isolated three overlapping cDNA clones encoding the E1 alpha subunit of the human enzyme complex. The composite human E1 alpha cDNA consists of 1783 base pairs encoding the entire human E1 alpha subunit of 400 amino acids with calculated Mr = 45,552. The human E1 alpha and the previously isolated human E2 cDNAs were used as probes in Northern blot analysis with cultured fibroblasts and lymphoblasts from seven unrelated MSUD patients. The results along with those of Western blotting have revealed five distinct molecular phenotypes according to mRNA and protein-subunit contents. These consist of type I, where the levels of E1 alpha mRNA and E1 alpha and E1 beta subunits are normal in cells, but E1 activity is deficient; Type II, where the E1 alpha mRNA is present in normal quantity, whereas the contents of E1 alpha and E1 beta subunits are reduced; Type III, where the level of E1 alpha mRNA is markedly reduced with a concomitant loss of E1 alpha and E1 beta subunits; Type IV, where the contents of both E2 mRNA and E2 subunits are markedly reduced; and Type V, where the E2 mRNA is normally expressed, but the E2 subunit is markedly reduced or completely absent. Type V includes thiamin-responsive (WG-34) and certain classical MSUD cells. These molecular phenotypes have demonstrated the complexity of MSUD and identified the affected gene in different patients for further characterization.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1995

Evidence for alternative splicing of the chicken vasoactive intestinal polypeptide gene transcript.

Two forms of chicken vasoactive intestinal polypeptide (VIP) mRNA have been identified by reverse transcription (RT)-PCR and RNase protection assay. The shorter form of chicken VIP mRNA encodes a protein that does not contain an analogue of rat peptide histidine isoleucine (PHI) 1-27 or human peptide histidine methionine 1-27. The larger form encodes both VIP and a chicken analogue of PHI 1-27 in the same protein product. Three VIP cDNAs isolated from a chicken hypothalamic cDNA library were derived from the shorter mRNA. Sequence analysis of the longest clone identified an open reading frame that codes for a 165 amino acid preproVIP protein and contains two polyadenylation signals. In situ hybridisation with an oligonucleotide probe from the VIP cDNA sequence showed that VIP-encoding mRNA occurs in cells in the basal hypothalamus, an area of the brain known to contain VIP neurosecretory neurones. RT-PCR of total RNA from liver, kidney, gut, pancreas, pituitary, cerebellum, forebrain and hypothalamus, using primers derived from the VIP cDNA sequence, showed that the shorter form of VIP mRNA is present in all of these tissues. The sequence of the longer form of VIP mRNA was obtained by sequencing a portion of the VIP gene from genomic DNA. This revealed a potential exon that was not represented in the VIP cDNA clones analysed. RT-PCR with primers from this sequence showed that it was expressed in the gut and hypothalamus. RNase protection assays confirmed the presence of the two forms of mRNA in gut and hypothalamus. The relative proportions of the two mRNA forms were: 97.8% VIP only, 2.2% PHI/VIP in the hypothalamus and 98.5% VIP only, 1.5% PHI/VIP in the gut. In conclusion, chicken VIP mRNA is alternatively spliced. The shortest form, which encodes a preproprotein containing only the VIP peptide, is the most abundant. The longer form of chicken VIP mRNA encodes a preproprotein containing sequences for both VIP and a chicken form of PHI.

Alternative Splicing↗
PubMed1990

A second thyroglobulin messenger RNA species (rTg-2) in rat thyrocytes.

A 0.95-kilobase (kb) thyrocyte RNA, initially detected in our 1B-6 subclone of Fisher rat thyrocytes (FRTL-5) using an oligonucleotide probe complementary to the 5' end of rat thyroglobulin (Tg) mRNA, was also detected in cultured thyrocytes of the Wistar rat (WRT cells) and in freshly isolated normal rat thyroid tissue. This transcript was thyroid specific and as abundant as the previously characterized 9.0-kb Tg mRNA in the cultured thyroid cells under the growth conditions employed. The smaller RNA (designated rTg-2 mRNA) was cytoplasmic, polyadenylated, and regulated by TSH. Preliminary characterization with several oligonucleotide probes showed that rTg-2 shared coding information present at the 5', but not the 3', end of the 9.0-kb Tg mRNA. Sequencing of the cloned rTg-2 cDNA showed that it was homologous to human and other higher vertebrate Tg cDNAs at its 5' coding end, but contained additional nonhomologous coding and noncoding sequences at the 3' end. The junction between the shared and unique sequences in rTg-2 mRNA occurred at the exon-5/intron-5 boundary in the Tg gene. This smaller transcript has not previously been reported and encodes elements known to be of structural and functional significance in the 330-kD Tg monomer. A putative polypeptide product from rTg-2 mRNA may play an important role in thyroid function and thyroid autoimmunity.

Amino Acid Sequence↗
PubMed1984

The nucleotide sequence of a complete chicken delta-crystallin cDNA.

The nucleotide sequence of a full length cDNA of delta-crystallin mRNA from chicken lens has been determined using a delta-crystallin cDNA clone (pB delta 11), which represents the mRNA sequence of 1530 nucleotides from the poly(A) junction but does not contain the 5'-terminal sequence of 44 nucleotides of the mRNA. The 5'-terminal sequence of the mRNA, absent in the cDNA clone, has been determined with a stretch of cDNA sequence by the primer extension procedure. The amino acid sequence deduced from the nucleotide sequence is consistent with the amino acid sequences of several tryptic peptides, the total amino acid composition, and the mol. wt. of delta-crystallin estimated by SDS-polyacrylamide gel electrophoresis. The computer-assisted analysis predicts high alpha-helical content throughout the polypeptide. Sequence analyses have revealed that gene 1 encodes the mRNA from which the cDNA clone was derived.

Amino Acid Sequence↗
PubMed1989

Multiple modifications in the phosphoproteins bound to stored messenger RNA in Xenopus oocytes.

Messenger RNA molecules accumulated in amphibian oocytes are stabilized and blocked from translation through association with a defined set of phosphoproteins. Phosphoproteins of 60 kDa and 56 kDa (pp60 and pp56) isolated from messenger ribonucleoprotein particles of Xenopus laevis oocytes can be bound in vitro to mRNA sequences. After phospholabelling in vitro, both pp60 and pp56 show a range of ionic forms, which resolve on two-dimensional gel electrophoresis as a series of pairs with identical charge. The similarities between pp60 and pp56 in their ionic properties suggest a common protein primary structure. This suggestion gains further support from proteinase digestion analysis of pp60 and pp56: practically identical size patterns of phospholabelled fragments are generated using a range of different proteinases. However, in spite of their structural similarities, pp60 and pp56 are recognised as antigenically distinct from each other by using polyclonal antibodies. It is concluded from these, and other, observations that pp60 and pp56 are members of a family of structurally similar polypeptides which are subjected to multiple secondary modifications. Of these modifications, phosphorylation appears to be instrumental in establishing tight binding to mRNA, while antigenicity appears to be determined by some other modification. The role of microheterogeneity in the structure of RNA-binding proteins is discussed in relation to the differential activation of mRNA sequences for translation during early development.

Animals↗
PubMed1989

Two species of the phosphoprotein mRNA with differently edited reading frames discovered in measles virus infected Vero cells. Brief report.

One cDNA clone representing the phosphoprotein mRNA sequence of the Edmonston strain of measles virus contained 4 G nucleotides at a particular position. Two other clones contained 3 G nucleotides at the same position. Otherwise the nucleotide sequence were identical. The mRNA with 3 G nucleotides codes for the 70 kD phosphoprotein. The mRNA with 4 G nucleotides may code for a putative new peptide with 231 aminoterminal amino acids in common with the P protein whereas the 68 carboxyterminal amino acids are different from any amino acid sequence of the phosphoprotein. Thus the consequence of the insertion of one additional G is a translational shift to a shorter open reading frame. There are several indications that the observation is of biological significance.

Animals↗
PubMed2004

Sequence and mRNA expression of nonclassical SLA class I genes SLA-7 and SLA-8.

Given the prominent position of pig endothelial cells in pig-to-human xenotransplantation and the role of classical and nonclassical MHC class I proteins in T and NK cell recognition, the expression of pig MHC (SLA) class I genes in a pig aortic endothelial cell line (AOC cells) was examined. Using a primer corresponding to a highly conserved region of exon 4, RT-PCR analysis of SLA class I expression in AOC cells revealed not only expression of the classical SLA class I ( SLA-1, -2, and -3) genes, but also SLA class I transcripts corresponding to SLA nonclassical class I (class Ib) genes SLA-6 and SLA-8. Further analysis of SLA class Ib expression in porcine aortic endothelial cells using SLA class I gene-specific primers confirmed SLA-6 and SLA-8 expression and also demonstrated expression of SLA-7. While SLA-6 has been relatively well characterized, no data regarding bona fide SLA-7 and SLA-8 transcripts have been reported. Therefore, cDNAs containing the complete open reading frames of SLA-6, -7, and -8 were obtained. Compared to an SLA-1 protein sequence, the predicted SLA-7 and -8 protein sequences exhibited most sequence divergence in alpha1, alpha2, and cytoplasmic domains. Expression of SLA-6, -7, and -8 was examined by RT-PCR using RNA prepared from a variety of tissues. SLA-6 transcripts were detected in every tissue examined. Except for brain, SLA-8 transcripts were similarly widespread. SLA-7 exhibited more limited tissue distribution.

Amino Acid Sequence↗
PubMed1994

The HNF-3 gene family of transcription factors in mice: gene structure, cDNA sequence, and mRNA distribution.

The rat HNF-3 (hepatocyte nuclear factor 3) gene family encodes three transcription factors known to be important in the regulation of gene expression in liver and lung. We have cloned and characterized the mouse genes and cDNAs for HNF-3 alpha, beta, and gamma and analyzed their expression patterns in various adult tissues and mouse embryonic stages. The HNF-3 proteins are highly conserved between mouse and rat, with the exception of the amino terminus of HNF-3 gamma, which in mouse is more similar to those of HNF-3 alpha and beta than to the amino termini of the rat HNF-3 gamma protein. The mouse HNF-3 genes are small and contain only two or three (HNF-3 beta) exons with conserved intron-exon boundaries. The proximal promoter of the mouse HNF-3 beta gene is remarkably similar to that of the previously cloned rat HNF-3 beta gene, but is different from the promoters of the HNF-3 alpha and gamma genes. The mRNA distribution of the mouse HNF-3 genes was analyzed by quantitative RNase protection with gene-specific probes. While HNF-3 alpha and beta are restricted mainly to endoderm-derived tissues (lung, liver, stomach, and small intestine), HNF-3 gamma is more extensively expressed, being present additionally in ovary, testis, heart, and adipose tissue, but missing from lung. Transcripts for HNF-3 beta and alpha are detected most abundantly in midgestation embryos (Day 9.5), while HNF-3 gamma expression peaks around Day 15.5 of gestation.

Amino Acid Sequence↗
PubMed1992

Homogeneously staining region in anthracycline-resistant HL-60/AR cells not associated with MDR1 amplification.

Anthracycline-resistant HL-60/AR cells and their drug-sensitive HL-60/S counterparts were characterized by karyotypic analysis and examined for the overexpression of DNA and mRNA sequences coding for P-glycoprotein (Pgp). The HL-60/S cells were karyotypically stable over a 5-year period of study (1986-1991), except for an additional small Giemsa-positive band noted at 7q22 in cultures harvested in 1987, but not in 1986. This change did not affect drug sensitivity. The drug-resistant HL-60/AR cells examined in 1986, 1987, and 1991 demonstrated a very stable karyotype. The most striking feature was a large homogeneously staining region in the long arm of chromosome 7 (7q11.2), and translocation of the remainder of the long arm to another centromere. Other changes in the HL-60/AR cells included inversion in 9q, partial deletion of the short arm of chromosome 10p, addition of material to the p arm of der(16), loss of chromosome 22, and the appearance of a new marker chromosome. Both HL-60/S and the HL-60/AR cells were found not to amplify DNA or mRNA sequences coding for the Pgp. Thus, although the HL-60/AR cells possess the classical multidrug resistance phenotype and demonstrate a homogeneously staining region near the region of the MDR1 gene, their resistance is due to mechanisms other than those coded for by MDR1.

Antibiotics, Antineoplastic↗
PubMed1986

Ontogenesis of growth hormone (GH)-responsive hepatic gene products: lack of correlation with hepatic GH receptor content.

The ontogenesis of hepatic GH receptor content is postulated as a major determinant of expression of hepatic GH-responsive gene products, since the ontogeny of the receptors and the responsive gene products, somatomedin-C, somatomedin-binding protein, and GH-responsive acidic protein, have similar qualitatively ontogenetic patterns. Two-dimensional gel electrophoresis of in vitro synthesized [35S]methionine-labeled proteins formed in the cell-free mRNA-dependent rabbit reticulocyte lysate system in response to hepatic RNA was used to quantitate the ontogenetic changes in a family of five GH-responsive gene products (S3, S11, S15, S16, and S20). These gene products were altered 2- to 12-fold by the administration of methionyl-human GH to adult hypophysectomized animals and were not altered by the combined administration of T4, corticosterone, and dihydrotestosterone. The translational activity of three of the five products (S3, S15, and S20) in animals younger than 15 days ranged from 1-12% of the values observed in normal adults and was consistent with diminished GH action. The translational activities of two mRNA sequences (S11 and S16) were not consistent with diminished GH action in the newborn animal. In the newborn animal, S11, induced 3-fold by GH in the adult animal, was 7.4 times the level observed in the adult. During the time of increasing GH receptor content (2-35 days), S16, attenuated by GH 2-fold in the adult, increased 6-fold. Injection of GH from days 2 through 11 augmented S20 and D2 (a hormonally nonresponsive, developmentally dependent product) 2-fold. The combined administration from days 2 through 11 of GH, T4, and corticosterone augmented S16 1.8-fold, a response paradoxical to that in the adult animal, but consistent with advancing maturation of the liver. We conclude that GH receptor content is not the sole determinant for the ontogenetic expression of GH-responsive products and that important alternate mechanisms exist for their regulation. In addition, GH, along with T4 and corticosterone, appear to modulate the rate at which adult levels of some mRNA sequences are achieved.

Animals↗
PubMed2006

The relation between mRNA folding and protein structure.

About 200 mRNA sequences of Escherichia coli and human with matching protein secondary structure data were studied. The mRNA folding for each native sequence and for corresponding randomized sequences was calculated through free energy minimization. We have found that the folding energy of mRNA segments in different protein secondary structures is significantly different. The average Z score is more negative for regular secondary structure (alpha-helix and beta-strand) than that for coil. This suggests that the codon choice in native mRNA sequence coding for protein regular structure contributes more to the mRNA folding stability.

Escherichia coli Proteins↗
PubMed1979

Reassessment of histone gene expression during cell cycle in human cells by using homologous H4 histone cDNA.

The representation of H4 histone mRNA sequences in RNAs isolated from G1 and S phase HeLa cells was assessed by use of a homologous H4 histone cDNA. S phase cells were obtained by double thymidine block, and G1 cells were obtained by double thymidine block or mitotic selective detachment. Nuclear and cytoplasmic RNAs from S phase cells hybridized with H4 histone cDNA as did nuclear and cytoplasmic RNAs from G1 cells synchronized by double thymidine block. In contrast, significant levels of hybridization were not observed between H4 histone cDNA and nuclear, polysomal, or postpolysomal cytoplasmic RNAs of G1 cells synchronized by mitotic selective detachment. Double thymidine block yields a G1 cell population containing 20-25% S phase cells whereas the G1 population obtained by mitotic detachment contains less than 0.1% S phase cells. The ability of H4 histone cDNA to hybridize with the RNAs from G1 cells obtained after release from double thymidine block can therefore be explained by the presence of S phase cells in such a G1 population--an artifact of the synchronization procedure. We interpret these results to be consistent with the presence of H4 histone mRNA sequences during the S but not G1 phase of the cell cycle in continuously dividing HeLa S3 cells.

Cell Cycle↗
PubMed2003

Messenger RNA editing in mammals: new members of the APOBEC family seeking roles in the family business.

Alteration of mRNA sequence through base modification mRNA editing frequently generates protein diversity. Several proteins have been identified as being similar to C-to-U mRNA editing enzymes based on their structural domains and the occurrence of a catalytic domain characteristic of cytidine deaminases. In light of the hypothesis that these proteins might represent novel mRNA editing systems that could affect proteome diversity, we consider their structure, expression and relevance to biomedically significant processes or pathologies.

APOBEC-1 Deaminase↗