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PubMedDate not supplied

[Cloning of double-stranded DNA--a transcript of rat transferrin mRNA].

Two-stage synthesis of double-stranded DNA was performed using purified rat transferrin mRNA as a template, reverse transcriptase and DNA polymerase I. Double-stranded transcripts of transferrin mRNA were cloned as recombinant plasmid derivatives of pBR322. The insert length in these plasmids varied from 150 to 1500 bp. Clones carrying transferrin mRNA sequences were identified using colony hybridization and Southern blot hybridization with 32P-cDNA probe. Nick-translated DNAs from transformed clones hybridized with a single component of rat liver polysomal RNA that corresponded to transferrin mRNA in its molecular weight (0.86 MD). In hybridization selection cell-free translation test cloned plasmid DNAs hybridized specifically with rat liver poly(A) +RNA that programmed the cell-free synthesis of a polypeptide identical to pretransferrin in antigenic properties and molecular weight.

Animals↗
PubMed2002

Many human genes are transcribed from the antisense promoter of L1 retrotransposon.

Human L1 retrotransposon has two transcription-regulatory regions: an internal or sense promoter driving transcription of the full-length L1, and an antisense promoter (ASP) driving transcription in the opposite direction into adjacent cellular sequences yielding chimeric transcripts. Both promoters are located in the 5'-untranslated region (5'-UTR) of L1. Chimeric transcripts derived from the L1 ASP are highly represented in expressed-sequence tag (EST) databases. Using a bioinformatics approach, we have characterized 10 chimeric ESTs (cESTs) derived from the EST division of GenBank. These cESTs contained 3' regions similar or identical to known cellular mRNA sequences. They were accurately spliced and preferentially expressed in tumor cell lines. Analysis of the hundreds of cESTs suggests that the L1 ASP-driven transcription is a common phenomenon not only for tumor cells but also for normal ones and may involve transcriptional interference or epigenetic control of different cellular genes.

5' Flanking Region↗
PubMed2004

Lead toxicity through the leadzyme.

Lead is one of the most dangerous toxic agents for all living organisms. In humans, elevated levels of lead have been linked to a number of disorders for which various molecular mechanisms have been proposed. However, none of them has been fully understood. It has also been known for several years that at micromolar concentrations lead can bind a unique RNA motif and catalyze a site-specific hydrolysis of the polyribonucleotide chain. This motif, called leadzyme, may be one of the major targets for lead within the cell, and it can cleave various cellular RNAs. A search of GenBank revealed the sequences that can potentially fold into the structure containing the leadzyme motif and that they are rather common in eukaryotic genomes. We found that the domain occurs with a high frequency in human mRNA sequences. Thus, the leadzyme nucleolytic properties should be considered as a possible mechanism for destruction of RNA within a cell. In particular, targeting of the RNA scaffold of ribosomes or spliceosomes may explain lead-mediated toxicity leading to cell death.

Humans↗
PubMed2002

Screening for antisense modulation of dystrophin pre-mRNA splicing.

Most gene therapy approaches to genetic disorders aim to compensate loss-of-function by introducing recombinant cDNA-based minigenes into diseased tissues. The current report represents an ongoing series of studies designed to correct genetic mutations at the post-transcriptional level. This strategy modifies the binding of components of the spliceosome by high affinity hybridisation of small complementary (antisense) RNA oligonucleotides to specific pre-mRNA sequences. These, so-called 'splicomer' reagents are chemically modified to impart bio-stability, and are designed to cause skipping of mutant frame-shifting exon sequences leading to restoration of the reading frame and an internally deleted but partially functional gene product. For instance, Duchenne muscular dystrophy is generally caused by frame-shift mutations in the dystrophin gene, whereas in-frame deletions of up to 50% of the central portion of the gene cause Becker muscular dystrophy, a much milder myopathy, which in some cases can remain asymptomatic to old age. In the mdx mouse model of Duchenne muscular dystrophy, a mutation in exon 23 of the dystrophin gene creates a stop codon and leads to a dystrophin-deficient myopathy in striated muscle. In previous studies, we have demonstrated that forced skipping of this mutant exon by treatment of mdx muscle cells with splicomer oligonucleotides can generate in-frame dystrophin transcripts and restore dystrophin expression. Here, we report the results of an optimisation of splicomer sequence design by the use of both high-throughput arrays and biological screens. This has resulted in specific and, importantly, exclusive skipping of the targeted exon in greater than 60% of dystrophin mRNA, leading to the de novo synthesis and localisation of dystrophin protein in cultured mdx muscle cells.

Animals↗
PubMed1998

Identification and characterization of a conserved erythroid-specific enhancer located in intron 8 of the human 5-aminolevulinate synthase 2 gene.

Thirty five kilobases of sequence encompassing the human erythroid 5-aminolevulinate synthase (ALAS2) gene have been determined. Analysis revealed a very low GC content, few repetitive elements, and evidence for the insertion of a reverse-transcribed mRNA sequence and a neighboring gene. We have investigated whether introns 1, 3, and 8, which correspond to DNase I-hypersensitivity sites in the structurally related mouse ALAS2 gene, affect expression of the human ALAS2 promoter in transient expression assays. Whereas intron 3 was marginally inhibitory, introns 1 and 8 of the human gene stimulated promoter activity. Intron 8 harbored a strong erythroid-specific enhancer activity which was orientation-dependent. Deletion analysis of this region localized enhancer activity to a fragment of 239 base pairs. Transcription factor binding sites clustered within this region include GATA motifs and CACCC boxes, critical regulatory sequences of many erythroid cell-expressed genes. These sites were also identified in the corresponding intron of both the murine and canine ALAS2 genes. Mutagenesis of these conserved sites in the human intron 8 sequence and transient expression analysis in erythroid cells established the functional importance of one GATA motif and two CACCC boxes. The GATA motif bound GATA-1 in vitro. The two functional CACCC boxes each bound Sp1 or a related protein in vitro, but binding of the erythroid Krüppel-like factor and the basic Krüppel-like factor could not be detected. The intron 8 enhancer region was not activated by GATA-1 together with Sp1 in transactivation experiments in COS-1 cells indicating the involvement of a related Sp1 protein or of another unidentified erythroid factor. Overall, these results demonstrate that a GATA-1-binding site and CACCC boxes located within the human ALAS2 intron 8 are critical for the erythroid-specific enhancer activity in transfected erythroid cells, and due to the conserved nature of these binding sites across species, it seems likely that these sites play a functional role in the tissue-restricted expression of the gene in vivo.

5-Aminolevulinate Synthetase↗
PubMed1993

Luteinizing hormone-releasing hormone gene expression in the bovine brain: anatomical localization and regulation by ovarian state.

Experiments were conducted to identify neurons in the bovine brain that express the LHRH gene and to determine whether LHRH mRNA levels are influenced by the ovaries. Two groups of postpubertal heifers were utilized: heifers killed during the mid-luteal phase of the estrous cycle (LUTEAL, n = 5) and heifers killed 14-16 wk following ovariectomy (OVX, n = 5). In situ hybridization was performed through use of a 32P-end-labeled deoxyoligonucleotide (59 mer) complementary to the human LHRH mRNA sequence. LHRH-expressing neurons were identified in the diagonal band of Broca, the preoptic area, and the anterior hypothalamus in a manner consistent with immunocytochemical localization. Reduced silver grains, proportional to LHRH mRNA content, were quantified (in pixels, 45x objective) with an image analysis system. Expected serum hormone concentration differences between endocrine states were confirmed by radioimmunoassay for progesterone (LUTEAL > OVX, p < 0.01) and for LH (OVX > LUTEAL, p < 0.01). Compared to the OVX group, LUTEAL heifers had 34% fewer LHRH-expressing neurons (p < 0.05); on the average, these neurons possessed 28% fewer pixels/cell (p < 0.01), indicating fewer copies of LHRH mRNA per cell. When the numbers of pixels in all labeled cells were totalled, LUTEAL animals had 57% fewer pixels (p < 0.05) than did the OVX females--probably reflecting a decrease in LHRH synthetic capacity in the LUTEAL animals. Therefore, during the mid-luteal phase of the bovine estrous cycle, ovarian steroid (i.e., luteal progesterone) suppression of LHRH release (as reflected by serum LH) is coincident with decreased LHRH mRNA in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Prognostic impact of cysteine proteases cathepsin B and cathepsin L in pancreatic adenocarcinoma.

OBJECTIVES: The cysteine proteases cathepsin B (CTSB) and L (CTSL) have been implicated in tumor spread and metastatic formation. In pancreatic adenocarcinoma, the role of these proteases is not very well defined. To find out which cell types produce CTSB and CTSL and to evaluate the prognostic impact of these proteases, 70 specimens from curatively resected patients with pancreatic adenocarcinoma were examined by in situ hybridization and immunohisto-chemistry. METHODS: Seventy patients with ductal adenocarcinoma of the pancreas were studied after R0 resection with a follow-up of at least 3 years. CTSB and CTSL expression was performed immunohisto-chemically using polyclonal anti-CTSB and CTSL antibodies. To detect cell types involved in producing CTSB and CTSL as well as the intracellular localization of specific mRNA sequences, nonisotopic in situ hybridization was performed. The correlations among CTSB and CTSL expression, clinicopathologic parameters, and clinical outcome were analyzed. RESULTS: The immunoreactivity was 96% for CTSB and 90% for CTSL. Positive mRNA signals were obtained in the cytoplasm tumor cells, macrophages, and fibroblasts in 77% for CTSB and 81% for CTSL, respectively. Statistical analysis showed a significant correlation between CTSB/CTSL expression and tumor grading (P < 0.05) and between CTSB and lymphatic invasion (P = 0.05). Kaplan-Meier analyses revealed statistical significance for CTSB/CTSL expression with the survival after curative resection (P < 0.05). Both proteases are strong prognostic markers in multivariate analysis (P = 0.0001) beside UICC stage, nodal status, tumor size, and grading (P < 0.05). Furthermore, CTSB expression is an independent prognostic marker for cancer recurrence within 6 months after curative surgery in multivariate analysis (P = 0.0001). CONCLUSIONS: CTSB and CTSL are strong and independent prognostic markers in resectable pancreatic adenocarcinoma rather than UICC stage, TNM classification, or tumor grading. Furthermore, CTSB is a predictor for early recurrence after curative resection. These data underline the significance of tumor-associated proteolysis for cancer invasion and metastasis and may lead to defining subgroups of patients with early recurrence and poor outcome.

Adult↗
PubMed2006

Differences in polyadenylation site choice between somatic and male germ cells.

BACKGROUND: We have previously noted that there were differences in somatic and male germ cell polyadenylation site choices. First, male germ cells showed a lower incidence of the sequence AAUAAA (an important element for somatic polyadenylation site choice) near the polyadenylation site choice. Second, the polyadenylation sites chosen in male germ cells tended to be nearer the 5' end of the mRNA than those chosen in somatic cells. Finally, a number of mRNAs used a different polyadenylation site in male germ cells than in somatic cells. These differences suggested that male germ cell-specific polyadenylation sites may be poor substrates for polyadenylation in somatic cells. We therefore hypothesized that male germ cell-specific polyadenylation sites would be inefficiently used in somatic cells. RESULTS: We tested whether pre-mRNA sequences surrounding male germ cell-specific polyadenylation sites (polyadenylation cassettes) could be used to direct polyadenylation efficiently in somatic cells. To do this, we developed a luciferase reporter system in which luciferase activity correlated with polyadenylation efficiency. We showed that in somatic cells, somatic polyadenylation cassettes were efficiently polyadenylated, while male germ cell-specific polyadenylation cassettes were not. We also developed a sensitive, 3' RACE-based assay to analyze polyadenylation site choice. Using this assay, we demonstrated that male germ cell-specific polyadenylation cassettes were not polyadenylated at the expected site in somatic cells, but rather at aberrant sites upstream of the sites used in male germ cells. Finally, mutation of the male germ cell-specific poly(A) signal to a somatic poly(A) signal resulted in more efficient polyadenylation in somatic cells. CONCLUSION: These data suggest that regulated polyadenylation site choice of male germ cell-specific polyadenylation sites requires one or more factors that are absent from somatic cells.

3T3 Cells↗
PubMed1978

Rapid accumulation of vitellogenin messenger RNA during secondary estrogen stimulation of Xenopus laevis.

Accurate quantitation of low concentrations of vitellogenin mRNA by hybridization to vitellogenin cDNA allows analysis of the accumulation of new vitellogenin mRNA sequences throughout secondary estrogen stimulation. Administration of a secondary injection of estradiol-17 beta to male Xenopus laevis which have been withdrawn from estrogen for 60 days results in synthesis of complete vitellogenin mRNA molecules in as little as 1 h after restimulation. Vitellogenin mRNA accumulates at a rate of 13 molecules/cell/min--at least four times the rate observed in primary estrogen stimulation and peaks at a level twice that observed in primary stimulation. Administration of estrogen to male Xenopus laevis evokes stable long lived changes in the pattern of vitellogenin gene expression and constitutes a type of cellular "memory effect."

Animals↗
PubMed1997

Alternative splicing of the human serotonin transporter gene.

To explore the structural basis for regulation of human serotonin transporter (hSERT) gene expression, we used primer extension and 5' rapid amplification of cDNA ends (5'RACE) techniques to estimate levels of and identify 5'-noncoding elements of hSERT mRNAs and genomic cloning to place these elements within the overall map of the hSERT gene. Primer extension on JAR cell mRNA suggested the presence of significant hSERT mRNA sequence upstream of the 5' end of our cloned hSERT cDNA. Using 5'RACE and reverse transcription-PCR (RT-PCR) methodologies, we cloned these sequences from brain and placenta and found this material to be composed of alternatively spliced exons using a previously reported noncoding exon (1A) and a novel 97-bp noncoding exon (1B). RT-PCR of JAR cell mRNA blotted with exon-specific oligonucleotides revealed both exons 1A and 1B to be regulated in a cholera toxin-dependent manner. To clarify the structure of the hSERT gene including exon 1B, we isolated and characterized genomic hSERT clones from Lambda Fix II and P1 artificial chromosome libraries. In agreement with previous findings, a single hSERT gene was identified that accounts for hybridizing bands on genomic Southern blots and was found to utilize 13 exons to encode the transporter's coding sequences along with the two noncoding 5' exons. Exon 1B was identified approximately 14 kb downstream of exon 1A in the hSERT gene and 737 bp upstream of exon 2, where the initiation site for translation is located. Exon 1B is surrounded by several elements potentially suitable for regulating serotonin transporter gene expression in vivo, including consensus sites for transcription factors AP-1, AP-2, CREB/ATF, and NF-kappaB. These data reveal additional complexity in hSERT gene structure and expression that may be relevant to regulated and compromised transporter expression in vivo.

Alternative Splicing↗
PubMed1980

Polyoma virus DNA: Sequence from the late region that specifies the leader sequence for late mRNA and codes for VP2, VP3, and the N-terminus of VP1.

The DNA sequence of part of the late region of the polyoma virus genome is presented. This sequence of 1,348 nucleotide pairs encompasses the leader region for late mRNA and the coding sequence for the two minor capsid proteins VP2 and VP3. The coding sequence for the N-terminus of the major capsid protein overlaps the C-terminus of VP2/VP3 by 32 nucleotide pairs. From the DNA sequence the sizes and sequences of VP2 and VP3 could be predicted. Potential splicing signals for the processing of late mRNA's could be identified. Comparisons are made between the sequence of polyoma virus DNA and corresponding regions of simian virus 40 DNA.

Amino Acid Sequence↗
PubMed2006

Genomic structure and an alternative transcript of bovine mitochondrial glycerol-3-phosphate acyltransferase gene (GPAM).

GPAM maps in BTA26q22, where several QTLs affecting milk production, milk fat and protein content have been mapped. On the basis of the QTL location, the GPAM gene could be considered a good candidate gene for the mentioned traits. Glycerol-3-phosphate acyltransferase mitochondrial (GPAM) is the enzyme that catalyses the initial and committed step of glycerolipid synthesis and, therefore, it is a potential site for triacylglycerol synthesis regulation. In this study, the structure of the cDNA and the genomic DNA of the bovine GPAM gene were determined and the expression of its mRNA was studied. The cDNA of the gene was cloned by RT-PCR, 5' and 3' rapid amplification of cDNA ends. The GPAM mRNA sequence contains a 2,475-bp coding region and a 3,689-bp 3' UTR. Its ORF encoded for an 825-amino acid protein and has an 89% homology with the coding regions of previously characterized mouse and human GPAM genes. The predicted amino acid sequence had an 89 and 93% similarity with mouse and human GPAM proteins, respectively. Using a 5' RACE strategy, two different 5' UTRs were cloned. Northern blot analysis confirmed the presence of two different transcripts. Adipose tissues and lung had the highest levels of GPAM mRNA expression, whereas it was barely detectable in liver. This expression pattern differs with those of non-ruminant animals where liver is one of the tissues with higher GPAM mRNA expression level.

3' Untranslated Regions↗
PubMed1992

The complementary deoxyribonucleic acid sequence of guinea pig endometrial prorelaxin.

The nucleotide sequence of the relaxin gene transcript in the endometrium of the late pregnant guinea pig has been determined. The strategy used was a combination of polymerase chain reaction (PCR) with primers designed from the mRNA sequence of porcine preprorelaxin, rapid amplification of cDNA ends-PCR, and blunt end cloning in M13 mp18. With heterologous primers, a 226-basepair (bp) segment of the guinea pig relaxin gene sequence was obtained and was used to design a guinea pig-specific primer for use with the rapid amplification of cDNA ends-PCR method. The latter allowed completion of the sequence of 336 bp, with a 96-bp overlap. The sequence obtained shows greater homology at both the nucleotide and amino acid levels with porcine and human relaxins H1 and H2 than with rat relaxin, supporting the thesis that the guinea pig is not a rodent. The transcription of the guinea pig endometrial relaxin gene during pregnancy was confirmed by Northern analysis of guinea pig endometrial tissues with a species-specific cDNA probe. The endometrial relaxin gene is transcribed during pregnancy, but not in lactation, consistent with the observed immunostaining for relaxin.

Amino Acid Sequence↗
PubMed1988

A Dictyostelium prespore-specific gene is transcriptionally repressed by DIF in vitro.

One important role of DIF, the stalk cell-specific inducer of Dictyostelium, may be to divert cells from the spore cell pathway of differentiation. The D19 gene encodes an mRNA which is highly enriched in prespore over prestalk cells in the migratory slug. We show, using a mutant defective in DIF accumulation, that the concentration of D19, and several other prespore mRNA sequences, decreases in the presence of exogenous DIF. There is evidence that both transcriptional and post-transcriptional controls operate to regulate expression of these genes. We have performed in vitro nuclear transcription and mRNA half-life analyses, and find that DIF acts at the transcriptional level to repress the accumulation of the D19 mRNA.

Cell Differentiation↗
PubMedDate not supplied

An O(N2) algorithm for discovering optimal Boolean pattern pairs.

We consider the problem of finding the optimal combination of string patterns, which characterizes a given set of strings that have a numeric attribute value assigned to each string. Pattern combinations are scored based on the correlation between their occurrences in the strings and the numeric attribute values. The aim is to find the combination of patterns which is best with respect to an appropriate scoring function. We present an O(N2) time algorithm for finding the optimal pair of substring patterns combined with Boolean functions, where N is the total length of the sequences. The algorithm looks for all possible Boolean combinations of the patterns, e.g., patterns of the form p and not q, which indicates that the pattern pair is considered to occur in a given string s, if p occurs in s, AND q does NOT occur in s. An efficient implementation using suffix arrays is presented, and we further show that the algorithm can be adapted to find the best k-pattern Boolean combination in O(Nk) time. The algorithm is applied to mRNA sequence data sets of moderate size combined with their turnover rates for the purpose of finding regulatory elements that cooperate, complement, or compete with each other in enhancing and/or silencing mRNA decay.

3' Untranslated Regions↗
PubMed1994

Expression of the c-myc protooncogene product in cells infected with the hepatitis delta virus.

The intrahepatic accumulation of the c-myc protooncogene product was observed on immunofluorescence in each of six patients with chronic hepatitis delta virus infection who exhibited the hepatitis D antigen in their livers. The c-myc product was stained in the same nuclei that contained the hepatitis D antigen. C-myc was not observed in acute hepatitis D or in cases of chronic hepatitis delta virus infection without expression of the hepatitis D antigen. The protooncogene product was detected in only 1 of 32 viral and nonviral liver disorders unrelated to hepatitis delta virus. To confirm these observations, we transfected HBsAg-positive (PCL/PRF/5) and HBsAg-negative (HepG2) transformed liver cell lines with a plasmid containing a hepatitis delta virus cDNA trimer under the control of the SV40 early enhancer/promoter sequences. Whereas baseline c-myc expression was barely detectable in mock-transfected PLC/PRF/5 or HepG2 cells, strong c-myc nuclear fluorescence was observed when these same cells were transfected with the hepatitis D antigen expression vector. Similar results were obtained after infection of HeLa cells with a recombinant vaccinia virus expressing the hepatitis D antigen. Detection of c-myc mRNA sequences by means of in situ hybridization suggested that the c-myc product accumulation was not due to increased amounts of its mRNA. The c-myc protein accumulates selectively in the livers of patients with chronic hepatitis delta virus infection and in the same nuclei that contain the hepatitis D antigen. The expression of c-myc in hepatitis D antigen-containing cells does not require the presence of hepatitis B virus infection.

Amino Acid Sequence↗
PubMed2003

Downregulation of bcl-2 expression in lymphoma cells by bcl-2 ARE-targeted modified, synthetic ribozyme.

Synthetic ribozymes are catalytic RNA molecules designed to inhibit gene expression by cleaving specific mRNA sequences. We investigated the potential of synthetic ribozymes to inhibit bcl-2 expression in apoptosis defective bcl-2 overexpressing tumors. A chemically stabilized hammerhead ribozyme has been targeted to the A+U-rich regulative element of bcl-2 mRNA that is involved in bcl-2 gene switch-off during apoptosis. The design of the ribozyme was based on the results of probing accessibility of the RNA target in cellular extracts with antisense DNA. The ribozyme was lipotransfected to a bcl-2 overexpressing human lymphoma cell line (Raji). The cellular uptake of this ribozyme resulted in a marked reduction of both bcl-2 mRNA and BCL-2 protein levels and dramatically increased cellular death by apoptosis. Our results suggest a potential therapeutic application of such ribozyme for the treatment of bcl-2 overexpressing tumors.

Apoptosis↗
PubMed2003

A novel serine protease of the mammalian HtrA family is up-regulated in mouse uterus coinciding with placentation.

This paper characterizes a novel gene, previously identified as uniquely regulated at implantation in mouse uterus. We cloned its full mRNA sequence encoding a serine protease possessing an IGF-binding domain and named it pregnancy-related serine protease (PRSP). PRSP is structurally similar to mammalian HtrA1 (56% amino acid similarity). Northern analysis revealed that the expression of PRSP mRNA was low before pregnancy, but it was increased at implantation and markedly up-regulated post-implantation. In-situ hybridization localized low levels of mRNA expression to the epithelium and stroma during very early pregnancy, but high expression to the decidual cells on day 8.5, primarily at the mesometrial pole where the placenta was forming. By day 10.5, PRSP mRNA was detected in the placenta. We also cloned an alternatively spliced PRSP mRNA that is expressed at a very low level. We located PRSP gene on chromosome 5 and established its intron/exon structure, which unambiguously explains how the two mRNA variants are produced through alternative splicing. Based on PRSP protein domain structure and its unique expression during pregnancy, we propose that PRSP plays an important role in the formation/function of the placenta.

Amino Acid Sequence↗