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PubMed1997

The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC.

The yeast splicing factor BBP (branchpoint bridging protein) interacts directly with pre-mRNA at or very near the highly conserved branchpoint sequence UACUAAC within the commitment complex. We also show that the recombinant protein recognizes the UACUAAC sequence. Therefore, BBP is also an acronym for branchpoint binding protein. The mammalian splicing factor SF1 is a BBP ortholog (mBBP) and an E complex component, and also has branchpoint sequence specificity. The relative conservation of this region in yeast and mammals correlates well with the RNA-binding differences between BBP and mBBP, suggesting that BBP contributes to branchpoint sequence definition in both systems.

Binding Sites↗
PubMed2000

Cloning, sequence and mRNA expression of bovine interleukin-16.

A lambda gt11 cDNA library was constructed using mRNA isolated from Theileria parva-infected bovine lymphocytes. Sequencing of random clones of this library resulted in the identification of a cDNA encoding bovine interleukin-16 (IL-16). The cDNA has an open reading frame of 1134 bp, and a 3' untranslated region of 275 nucleotides with a polyadenylation signal 16 bases upstream from the poly (A) tail. The protein predicted by the cDNA sequence contains 378 amino acids and the level of amino acid homology with the corresponding part of human precursor IL-16 is 79 %. No information is available about the tissue distribution of IL-16 in cattle, therefore we investigated the expression of IL-16 mRNA in bovine lymphoid tissues by reverse-transcription polymerase chain reaction assays. To investigate the potential of IL-16 as an immunoregulatory molecule we also analysed IL-16 mRNA expression in CD4+ and CD8+T-cell clones derived from T. parva-immunised cattle.

Amino Acid Sequence↗
PubMed1985

A lethal deletion on mouse chromosome 7 affects regulation of liver-cell-specific functions: posttranscriptional control of serum protein and transcriptional control of aldolase B synthesis.

Steady-state levels of mRNAs were determined for the serum proteins albumin, alpha-fetoprotein (AFP), and transferrin, as well as for aldolase B in livers of newborn mice homozygous for a radiation-induced lethal deletion (c14CoS) in chromosome 7. Deficiencies in synthesis and secretion of the serum proteins as well as in activities of certain liver-specific enzymes characterize these homozygotes. The results of RNA dot and gel-blot hybridizations with the respective cloned cDNA probes showed a decrease to one-fourth of aldolase B mRNA levels in homozygous mutant livers compared to normal littermates, in contrast to normal levels of mRNA sequences for the three serum proteins in the mutants. Furthermore, the mRNA sequences were shown to be present as mature mRNA molecules in both mutant and normal littermate livers. We suggest that the deficiencies of liver-specific serum proteins and those of the enzymes caused by the lethal deletions around the albino locus on chromosome 7 of the mouse are due to different causes. In the case of the liver-specific enzyme examined here--i.e., aldolase B--control at the level of transcription or of message stability is affected in the homozygous deletion mutants, whereas the deficiencies of serum proteins are not reflected on the mRNA level and owe their origin to an effect on a posttranscriptional or translational level. These results lend further support to the assumption that the deleted portion of the genome includes genes concerned with the control and regulation of liver cell differentiation.

Animals↗
PubMed1978

Regulation of prolactin and somatotropin mRNAs by thyroliberin.

The tripeptide thyroliberin (thyrotropin-releasing hormone, TRH) regulates the levels of cytoplasmic prolactin and growth hormone (somatotropin) mRNAs in a line of functional rat pituitary tumor cells. Intracellular mRNA levels were determined with specific complementary DNA probes in RNA-excess hybridization reactions and independently by two-dimensional gel electrophoretic analysis of the products of mRNA-directed cell-free translation. Prolactin mRNA sequences increased from 1.1 to 4.5% of the cytoplasmic poly(A)containing mRNA within 48 hr after addition of TRH to the medium; in parallel, growth hormone mRNA sequences were decreased. Similar effects on prolactin mRNA levels were observed in cells grown in serum-containing medium or when the cells were maintained in a serum-free medium for 72 hr prior to addition of TRH, suggesting that TRH is responsible for the stimulation of prolactin mRNA levels rather than for exerting a permissive effect for another factor present in serum.

Cell Line↗
PubMed1983

Hormonal regulation of plasminogen activator mRNA production in porcine kidney cells.

Plasminogen activator (PA) production in LLC-PK1 pig kidney cell culture is induced to high levels by calcitonin and vasopressin, both of which stimulate adenylate cyclase, or by other compounds that also raise intracellular cAMP levels. Enzyme induction is transiently sensitive to inhibition by actinomycin D, suggesting that increased concentrations of cAMP mediate the inducing effects of the hormones by enhancing the transcription of PA-mRNA sequences. We tested this hypothesis by measuring PA-mRNA sequences in the Xenopus oocyte translation system which showed a 15-20-fold enhanced PA-synthesizing capacity when supplied with poly(A)+RNA from induced cells, above that obtained from uninduced cell RNA. Changes in PA-mRNA levels measured by Northern hybridization using cloned PA-specific cDNA gave results that agreed well with those obtained from translation assays. Pretreatment with high concentrations of cycloheximide did not block calcitonin-induced PA-mRNA synthesis, indicating that PA gene activation was a primary transcriptional result of calcitonin stimulation and did not require new protein synthesis.

Animals↗
PubMed1992

Lethal perinatal osteogenesis imperfecta due to a type I collagen alpha 2(I) Gly to Arg substitution detected by chemical cleavage of an mRNA:cDNA sequence mismatch.

A single base mismatch was detected by a chemical cleavage method in heteroduplexes formed between patient mRNA and a control collagen alpha 2(I) cDNA probe in a case of osteogenesis imperfecta type II. The region of the mRNA mismatch was amplified using the polymerase chain reaction, cloned and sequenced. A heterozygous point mutation of G to C at base pair 1,774 of the collagen alpha 2(I) mRNA resulted in the substitution of glycine with arginine at amino acid position 457 of the helix. Type I collagen of alpha 1(I)- and alpha 2(I)-chains from the patient migrated slowly on electrophoresis due to increased levels of posttranslational modification of lysine. The parents' fibroblast collagen did not contain the mRNA mismatch and the collagens showed normal electrophoretic behaviour. Two-dimensional electrophoresis of the CNBr peptides from the patient's collagen confirmed the excessive posttranslational modification of the alpha 1(I)- and alpha 2(I)-chains in the CNBr peptides N-terminal to the mutation due to disruption of the obligatory Gly-X-Y triplet repeat of the helix. The mutation led to reduced procollagen secretion and helix destabilization as evidenced by a decreased thermal stability. These data lend further support to the accumulating evidence that type I collagen alpha 2(I) glycine substitution mutations result in the same spectrum of clinical severity as those in the alpha 1(I)-chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1981

Pancreas-specific genes: structure and expression.

Via recombinant DNA technology the mRNA sequence of pancreatic amylase has been cloned and its nucleotide sequence has been determined. The cloned sequence represents 96% of the total length of amylase mRNA; missing are an estimated 75 +/- 30 nucleotides from the 5' end. The amino acid sequence of rat pancreatic amylase was deduced solely from the nucleotide sequence of the mRNA. Unlike other eukaryotic mRNAs, the amylase mRNA has short 5' and 5' untranslated regions, suggesting that long untranslated regions of eukaryotic mRNAs either do not contain extensive functional sequences or that these sequences are incorporated within the amino acid coding region of amylase mRNA. The cloned amylase mRNA sequence was radiolabeled and used as a probe for in situ hybridization. These experiments demonstrate that amylase mRNA is present in all acinar cells but not in other pancreatic cell types. Using the cloned amylase mRNA sequences as a hybridization probe, three nonoverlapping genomic DNA fragments containing amylase gene sequences were isolated. From the similar sequence organization of the three amylase genes visualized by DNA heteroduplex mapping, a consensus structure of a rat amylase gene is proposed. It is an extended gene structure 10 kilobase pairs in length containing the 1547 base pairs of the cloned mRNA coding sequence interrupted by seven intervening sequences ranging from 400-2000 base pairs long. Thus, in nuclear DNA the amylase mRNA coding sequence is disrupted into at least eight segments from 150-300 base pairs long.

Amino Acid Sequence↗
PubMed1998

EDEN and EDEN-BP, a cis element and an associated factor that mediate sequence-specific mRNA deadenylation in Xenopus embryos.

During Xenopus early development, gene expression is regulated mainly at the translational level by the length of the poly(A) tail of mRNAs. The Eg family and c-mos maternal mRNAs are deadenylated rapidly and translationally repressed after fertilization. Here, we characterize a short sequence element (EDEN) responsible for the rapid deadenylation of Eg5 mRNA. Determining the core EDEN sequence permitted us to localize the c-mos EDEN sequence. The c-mos EDEN confered a rapid deadenylation to a reporter gene. The EDEN-specific RNA-binding protein (EDEN-BP) was purified and a cDNA obtained. EDEN-BP is highly homologous to a human protein possibly involved in myotonic dystrophy. Immunodepleting EDEN-BP from an egg extract totally abolished the EDEN-mediated deadenylation activity, but did not affect the default deadenylation activity. Therefore, EDEN-BP constitutes the first trans-acting factor for which an essential role in the specificity of mRNA deadenylation has been directly demonstrated.

Adenine↗
PubMed1983

Synthesis and processing of simian virus 40-specific RNA in adenovirus-infected, simian virus 40-transformed human cells.

Human simian virus 80 (SV80) cells transformed by simian virus 40 (SV40) synthesize substantial quantities of the SV40 large T-antigen (Henderson & Livingston, 1974; Tjian, 1978) and cytoplasmic, poly(A)-containing RNA species that exhibit spliced structures characteristic of the SV40, early messenger RNA species that encode both large and small T-antigens (Flint & Beltz, 1979). When SV80 cells were infected with type C adenovirus, both the synthesis of SV40 large T-antigen and the appearance in the cytoplasm of newly synthesized, SV40-specific RNA sequences were inhibited during the late phase of infection. The results of hybridization to SV40 DNA of SV80 nuclear RNA, prepared from mock- or adenovirus-infected cells after labeling for short periods in vivo or in vitro, indicated that transcription of integrated SV40 was, by contrast, not disrupted during the late phase of adenovirus infection. Poly(A)-containing, nuclear RNA species that hybridized to SV40 DNA sequences and exhibited the sizes of spliced, large and small T-antigen mRNA species were also synthesized in infected cells at a time when the corresponding mRNA sequences did not leave the nucleus. These results suggest that the failure of non-adenoviral mRNA sequences to enter the cytoplasm of adenovirus-infected cells does not reflect inhibition of either their transcription or the normal enzymatic processing reactions to which pre-mRNA species are subject. Several lines of evidence do, however, establish that nuclear, SV40-specific RNA sequences are less stable in adenovirus-infected compared to mock-infected SV80 cells.

Adenoviruses, Human↗
PubMed1984

Computer programs used to aid in the selection of DNA hybridization probes.

This paper describes a package of three programs which used together aid in selecting the best possible sequence to be used as a DNA hybridization probe. This system searches an amino acid sequence for four adjacent amino acids with the fewest possible corresponding mRNA sequences, calculates their probability of occurrence, and locates the positions of wobbles and mismatches between the DNA hybridization probe and the possible mRNA sequences.

Base Sequence↗
PubMed2005

Molecular cloning and characterization of beta-defensin cDNA expressed in distal ileum of buffalo (Bubalus bubalis).

Defensins play a prominent role in protection of various epithelial surfaces. In this study, we have cloned and characterized the mRNA from the distal ileum of Bubalus bubalis. Total RNA after isolation from ileal epithelium was reverse transcribed to synthesize cDNA using primers designed by taking conserved region of cattle enteric beta-defensin (EBD) mRNA, goat beta-defensin 2 (BD 2) and cattle lingual antimicrobial peptide (LAP) mRNA sequences. The PCR amplified cDNA of 254 bp was ligated to pDrive cloning vector and transformed into XL-blue strain of E coli. The sequence analysis indicated 29 nucleotide substitutions with reported cattle EBD mRNA sequence sharing 86.2% homology, 92.1% with cattle LAP, 81.6% with cattle tracheal antimicrobial peptide and 84.6% with goat BD 2. The deduced amino acid sequence encodes for a 64 amino acid precursor peptide. Both nucleotide and amino acid sequence homology shows that the cloned sequence is closer to cattle LAP.

Amino Acid Sequence↗
PubMed1978

In vitro RNA synthesis and expression of vitellogenin gene in isolated chicken liver nuclei.

Optimal conditions for prolonged in vitro synthesis of RNA in isolated chicken liver nuclei have been described. It is shown by incorporation of gamma32P-GTP into RNA, analysis of the product on sucrose density gradient, and digestion with alkaline phosphatase and ribonuclease A that there is reinitiation of RNA synthesis. Polynucleotide kinase activity has been ruled out as explanation for the incorporation of gamma32P-GTP. alpha-Amanitin inhibits RNA synthesis by about 50%. Nuclei prepared from estradiol-treated chicks have twice the RNA synthesis activity as the controls. RNA is synthesized in the presence of Hg-UTP and the mercurated product separated by affinity chromatography on sulfhydryl-Sepharose column under stringent conditions. Vitellogenin mRNA sequences are measured by hybridization with DNA complementary to vitellogenin mRNA. Estradiol treatment leads to a 10-fold increase in vitellogenin mRNA sequences.

Amanitins↗
PubMed1981

Hormonal modulation of alpha-fetoprotein gene expression in newborn rat livers.

Suppression of serum alpha-fetoprotein (AFP) levels in glucocorticoid treated newborn rats was investigated. Daily intraperitoneal injection of 2 micrograms/g body weight of dexamethasone into newborn rats greatly reduced the concentration of AFP in the serum and liver cytosol. In contrast, this treatment stimulated liver ornithine decarboxylase activity. The reduction in AFP levels is not due to a change of distribution of AFP molecular variants, inhibition of secretion of synthesized AFP by the liver or disruption of liver polysomes. Glucocorticoids decrease the AFP levels in hormone-treated rats by supressing the synthesis of AFP. The size of AFP polysomes isolated from the livers of dexamethasone-treated rats were as large as those from normal rats. However, the amount of AFP-producing polysomes in hormone-treated rat liver is only 14% of the controls. By hybridization assays, it was found that dexamethasone treated livers contained decreased amounts of AFP mRNA sequences in liver cytoplasmic and nuclear RNAs. The decreased amounts of AFP mRNA sequences in hormone-treated liver are caused by both a decrease in the rate of AFP mRNA transcription and in AFP mRNA stability.

Animals↗
PubMed1995

Adhalin mRNA and cDNA sequence are normal in the cardiomyopathic hamster.

Adhalin is deficient in two forms of human muscular dystrophy, one due to mutations in the adhalin gene and one linked to an unidentified gene on chromosome 13. Because adhalin is deficient in skeletal and cardiac muscles of BIO 14.6 hamsters, which experience both myopathy and cardiomyopathy, cDNA encoding adhalin from BIO 14.6 hamster skeletal muscle was cloned and sequenced. Adhalin mRNA was expressed at normal levels in BIO 14.6 hamster cardiac muscle, and no mutation in adhalin coding sequence was found, indicating that the inherited myopathy and cardiomyopathy of the BIO 14.6 hamster are most likely not due to mutations in the adhalin gene.

Amino Acid Sequence↗
PubMed2003

The Krüppel-like factor epiprofin is expressed by epithelium of developing teeth, hair follicles, and limb buds and promotes cell proliferation.

We identified a cDNA clone for epiprofin, which is preferentially expressed in teeth, by differential hybridization using DNA microarrays from an embryonic day 19.5 mouse molar cDNA library. Sequence analysis revealed that this cDNA encodes a member of the Krüppel-like factor family containing three characteristic C2H2-type zinc finger motifs. The full-length cDNA was obtained by the 5' Cap capture method. Except for its 5'-terminal sequence, the epiprofin mRNA sequence is almost identical to the predicted sequence of Krüppel-like factor 14/Sp6 (specificity protein 6), which was previously identified in expressed sequence tag data bases and GenBank by an Sp1 zinc finger DNA-binding domain search (Scohy, S., Gabant, P., Van Reeth, T., Hertveldt, V., Dreze, P. L., Van Vooren, P., Riviere, M., Szpirer, J., and Szpirer, C. (2000) Genomics 70, 93-101). This sequence difference is due to differences in the assignment of the location of exon 1. In situ hybridization revealed that epiprofin mRNA is expressed by proliferating dental epithelium, differentiated odontoblast, and also hair follicle matrix epithelium. In addition, whole mount in situ hybridization showed transient expression of epiprofin mRNA in cells of the apical ectodermal ridge in developing limbs and the posterior neuropore. Transfection of an epiprofin expression vector revealed that this molecule is localized in the nucleus and promotes cell proliferation. Thus, epiprofin is a highly cell- and tissue-specific nuclear protein expressed primarily by proliferating epithelial cells of teeth, hair follicles, and limbs that may function in the development of these tissues by regulating cell growth.

Amino Acid Sequence↗
PubMed2005

Stemless self-quenching reporter molecules identify target sequences in mRNA.

The design of oligonucleotides for gene silencing requires a rational method for identifying hybridization-accessible sequences within the target RNA. To this end, we have developed stem-loop self-quenching reporter molecules (SQRMs) as probes for such sequence. SQRMs have a 5' fluorophore, a quenching moiety on the 3' end, an intervening sequence that forms an approximately 5-basepaired stem, and a loop sequence of approximately 20-30 bases. We have previously described a mapping strategy employing SQRMs to locate stem-loop structures in the target mRNA molecule. We now show that the original design constraint of a basepaired stem is not needed, either in vitro or in vivo. We propose that stemless probes possess sufficient signal-to-noise for use in vivo and that this ratio is an indication of hybridization of the probe to its target. Data showing that these SQRMs can specifically target and reduce c-Myb protein synthesis and can be used for real-time in vivo assays are presented.

Animals↗
PubMed1995

cDNA sequence and mRNA tissue distribution of a novel human matrix metalloproteinase with a potential transmembrane segment.

The complementary DNA sequence of a novel matrix metalloproteinase was isolated from a human lung cDNA library. It consists of 3530 bp and encodes a polypeptide of 669 amino acids. In comparison to other matrix metalloproteinases, the deduced sequence of the amino acid chain exhibits closest similarity to a recently discovered membrane-type matrix metalloproteinase of 582 amino acids. Likewise, it is composed of a signal peptide, a prodomain, a catalytic domain, a hemopexin-homologous domain and a C-terminal domain. Furthermore, the novel matrix metalloproteinase shares a similar activation site with its 582-amino-acid homologue, an insertion of eight amino acids in the catalytic domain and a tract of more than 20 hydrophobic amino acids near the C-terminus. The hydrophobic structure in the C-terminal domain suggests that the novel matrix metalloproteinase is also membrane bound. When lung cell membrane fractions were probed in immunoblots with polyclonal antibodies against a recombinant fragment of the 669-amino-acid chain, a protein of M(r) 72,000 reacted preferentially with the antibodies. Northern-blot analysis demonstrated quite different tissue distributions of mRNA for the two membrane-type matrix metalloproteinases. While mRNA for the 582-amino-acid enzyme was found predominantly in lung, placenta, kidney, ovary, intestine, prostate and spleen, mRNA for the 669-amino-acid enzyme appeared to be synthesized preferentially in liver, placenta, testis, colon and intestine. Substantial amounts of the latter mRNA were also detected in pancreas, kidney, lung, heart and skeletal muscle.

Amino Acid Sequence↗
PubMed1983

Cloning and characterization of the rat NADPH-cytochrome P-450 oxidoreductase gene.

A rat liver DNA genomic library was prepared using the lambda phage cloning vector Charon 28. Recombinant phage were screened with a cDNA clone (pOR-7) containing sequences complementary to mRNA coding for NADPH-cytochrome P-450 oxidoreductase. This cDNA clone contains the poly(A) addition site and 60% of the mRNA sequence (Gonzalez, F. J., and Kasper, C. B. (1982) J. Biol. Chem. 257, 5962-5968). Four positive phage were identifed and plaque-purified, and their DNA was isolated and subjected to restriction endonuclease mapping. All phage DNA inserts, which ranged from 11 to 16 kilobases, contained several overlapping restriction fragments. The clone with the largest insert (lambda OR-2) was found to contain restriction fragments identical with those of rat DNA when both were subjected to Southern blotting with nick-translated pOR-7 DNA; this finding established the presence in lambda OR-2 of the 3' end (poly(A) addition site) of the oxidoreductase gene. When [32P]cDNA synthesized from enriched oxidoreductase poly(A) RNA was utilized as a probe, additional fragments were identified. The fragment most distal to the 3'-specific fragment was assumed to contain the 5' cap site and was subcloned into pBR322 for further analysis. Restriction mapping and Southern blot analysis further localized the 5' end of the gene to an AvaII fragment of 540 base pairs (bp). Hybridization of this fragment with oxidoreductase mRNA-enriched poly(A) RNA resulted in the arrest of translation of oxidoreductase; this confirmed that it contained an exon region of the oxidoreductase gene. S1 nuclease mapping and DNA sequencing identified to within +/- 1 bp the 5' cap site of the gene which corresponds to an A start. DNA sequencing of the 5'p flanking region revealed no "TATA box" in the vicinity of -25 to -30 bp of the cap site. R-loop analysis of lambda OR-2 revealed the presence of a minimum of seven introns in the 6000-bp oxidoreductase gene and eight exons with a total length of approximately 2600 bp.

Animals↗