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PubMed2004

RNase P ribozyme inhibits cytomegalovirus replication by blocking the expression of viral capsid proteins.

By linking a guide sequence to the catalytic RNA subunit of RNase P (M1 RNA), we constructed a functional ribozyme (M1GS RNA) that targets the overlapping mRNA region of two human cytomegalovirus (HCMV) capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, a reduction of >85% in the expression of CSP and assemblin and a reduction of 4000-fold in viral growth were observed in the HCMV-infected cells that expressed the functional ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in virus-infected cells that either did not express the ribozyme or produced a 'disabled' ribozyme carrying mutations that abolished its catalytic activity. Characterization of the effects of the ribozyme on the HCMV lytic replication cycle further indicates that the expression of the functional ribozyme specifically inhibits the expression of CSP and assemblin, and consequently blocks viral capsid formation and growth. Our results provide the direct evidence that RNase P ribozymes can be used as an effective gene-targeting agent for antiviral applications, including abolishing HCMV growth by blocking the expression of the virus-encoded capsid proteins.

Animals↗
PubMed1996

Expression of major surface protein 2 antigenic variants during acute Anaplasma marginale rickettsemia.

Antigenic variants of Anaplasma marginale major surface protein 2 (MSP-2), a target of protective immune responses, have been detected by use of copy-specific monoclonal antibodies reactive with some, but not all, organisms during acute rickettsemia. The presence of polymorphic msp-2 genes was confirmed by cloning and sequencing two gene copies, 11.2 and DF5, each of which encodes a full-length MSP-2 with a unique amino acid sequence. Transcription of msp-2 genes during acute rickettsemia was analyzed by use of cDNA cloning of hybrid-selected msp-2 mRNA. Sequencing of cDNA clones, designated AR1 to AR14, indicated that DF5 msp-2 was transcribed during acute rickettsemia. Two classes of variant msp-2 genes were also transcribed during acute rickettsemia. The first class of variant transcripts, typified by clones AR3, AR4, AR7, and AR14, each encoded a single or small number of amino acid substitutions relative to DF5. The second type, AR5, encoded a large region of amino acid polymorphism, including additions, deletions, and substitutions, as compared to DF5. Specific antibody directed against the AR5 polymorphic region bound a unique MSP-2 expressed on A. marginale that was not recognized by antibody generated against DF5. Similarly, anti-AR5 peptide antibody reacted with a different MSP-2 that was not bound by anti-DF5 antibody. This expression confirmed that variant msp-2 transcripts encode structurally distinct MSP-2 molecules which bear unique B-cell epitopes. These results support the hypothesis that the large msp-2 gene family, which constitutes a minimum of 1% of the genome, encodes antigenic variants critical to evasion of protective immune response directed against surface MSP-2 epitopes.

Acute Disease↗
PubMed2000

Use of serial analysis of gene expression to generate kidney expression libraries.

Chronic renal disease initiation and progression remain incompletely understood. Genome-wide expression monitoring should clarify mechanisms that cause progressive renal disease by determining how clusters of genes coordinately change their activity. Serial analysis of gene expression (SAGE) is a technique of expression profiling, which permits simultaneous, comparative, and quantitative analysis of gene-specific, 9- to 13-bp sequence tags. Using SAGE, we have constructed a tag expression library from ROP-+/+ mouse kidney. Tag sequences were sorted by abundance, and identity was determined by sequence homology searching. Analyses of 3,868 tags yielded 1,453 unique kidney transcripts. Forty-two percent of these transcripts matched mRNA sequence entries with known function, 35% of the transcripts corresponded to expressed sequence tag (EST) entries or cloned genes, whose function has not been established, and 23% represented unidentified genes. Previously characterized transcripts were clustered into functional groups, and those encoding metabolic enzymes, plasma membrane proteins (transporters/receptors), and ribosomal proteins were most abundant (39, 14, and 12% of known transcripts, respectively). The most common, kidney-specific transcripts were kidney androgen-regulated protein (4% of all transcripts), sodium-phosphate cotransporter (0.3%), renal cytochrome P-450 (0.3%), parathyroid hormone receptor (0.1%), and kidney-specific cadherin (0.1%). Comprehensively characterizing and contrasting gene expression patterns in normal and diseased kidneys will provide an alternative strategy to identify candidate pathways, which regulate nephropathy susceptibility and progression, and novel targets for therapeutic intervention.

Animals↗
PubMed1986

Cloning and sequence analysis of a partial cDNA for chicken cartilage proteoglycan core protein.

A chicken embryo sternal cartilage cDNA library, created in the plasmid expression vector pUC9, was screened for sequences coding for immunologically detectable core protein of the large, major proteoglycan of cartilage. A 1229-base-pair cDNA clone was isolated that contained only one extended open reading frame, which had sequences coding for a polypeptide of 379 amino acid residues. These deduced sequences corresponded to those anticipated from current models of proteoglycan structure; a deduced sequence encompassing 21 amino acids was almost identical to a known sequence of bovine nasal cartilage proteoglycan. Significant homology was found between the deduced amino acid sequence of the proteoglycan and two regions of a chicken hepatic lectin. Immunoprecipitation of the products of cell-free translation yielded a component of about 340 kDa, and transfer blot hybridization of sternal cartilage RNA showed a single mRNA of about 8.1 kilobases. Hybridizable mRNA sequences were readily detectable by dot-blot analyses of the cytoplasm of cartilaginous tissues of the chicken embryo, whereas similar analyses of prechondrogenic limb mesenchymal cells did not demonstrate such hybridizable mRNA signals.

Amino Acid Sequence↗
PubMed1996

Reverse transcriptase/polymerase chain reaction detection of cytokeratin-19 mRNA in bone marrow and blood of breast cancer patients.

A two-step reverse-transcriptase-based polymerase chain reaction (PCR) with nested primer pairs was developed to amplify sensitive and specific cytokeratin-19 (CK-19) mRNA sequences from human breast cancer cells. No CK-19 pseudogene interference was seen. The larger DNA-derived amplification products could be clearly discriminated from mRNA-derived products. The CK-19 message was not amplified from bone marrow or blood of healthy volunteers and patients with haematological malignancies nor from myeloid and lymphoid cell lines. Breast cancer cells were diluted in buffy coat cells up to 10(-6) and CK-19 mRNA sought by PCR. The CK-19 message was detected in 14 of 26 blood samples and 14 of 24 marrow samples but in neither of two peripheral blood stem cell samples taken from 35 breast cancer patients. By sequence-analysis control of two of these samples and two cell lines, the amplified DNA fragments were confirmed to be homologous with the CK-19 sequence. The CK-19 message was further sought in matched blood/marrow samples taken from 13 untreated women in the same cohort at the time of diagnosis. In 3 of these, CK-19 RNA was detected in blood and marrow and, in 3 others, only in blood, but never in marrow alone. The results show that CK-19 assay by reverse transcriptase/PCR is a sensitive and specific technique for the detection of cancer cells in bone marrow and blood. It could be helpful in diagnosis and monitoring of metastatic breast cancer and detection of micrometastases. This should be evaluated on larger numbers of patients, with different clinical samples and epithelial malignancies.

Base Sequence↗
PubMed2006

RANDNA: a random DNA sequence generator.

Monte Carlo simulations are useful to verify the significance of data. Genomic regularities, such as the nucleotide correlations or the not uniform distribution of the motifs throughout genomic or mature mRNA sequences, exist and their significance can be checked by means of the Monte Carlo test. The test needs good quality random sequences in order to work, moreover they should have the same nucleotide distribution as the sequences in which the regularities have been found. Random DNA sequences are also useful to estimate the background score of an alignment, that is a threshold below which the resulting score is merely due to chance. We have developed RANDNA, a free software which allows to produce random DNA or RNA sequences setting both their length and the percentage of nucleotide composition. Sequences having the same nucleotide distribution of exonic, intronic or intergenic sequences can be generated. Its graphic interface makes it possible to easily set the parameters that characterize the sequences being produced and saved in a text format file. The pseudo-random number generator function of Borland Delphi 6 is used, since it guarantees a good randomness, a long cycle length and a high speed. We have checked the quality of sequences generated by the software, by means of well-known tests, both by themselves and versus genuine random sequences. We show the good quality of the generated sequences. The software, complete with examples and documentation, is freely available to users from: http://www.introni.it/en/software.

Base Sequence↗
PubMed1989

Deduced sequence of a malate synthase polypeptide encoded by a subclass of the gene family.

We analyzed five malate synthase cDNA clones from the higher plant Brassica napus L. We determined the complete mRNA sequence and showed that the longest cDNA clone, pMS1, contains the entire protein coding region. The deduced polypeptide consists of 561 amino acids with a molecular mass of 63,700 daltons. To discern whether the cloned mRNAs represent distinct malate synthase polypeptides, we compared restriction maps and partial nucleotide sequence of the cDNA clones as well as their pattern of hybridization with restriction fragments in nuclear DNA. The results suggest that the five cloned mRNAs are encoded by either a single gene or by highly conserved members of the gene family.

Amino Acid Sequence↗
PubMed1989

Characterization of a cDNA for rat P-450g, a highly polymorphic, male-specific cytochrome in the P-450IIC subfamily.

Cytochrome P-450g (IIC13) is a highly polymorphic, male-specific rat liver isozyme which is a member of the P-450IIC subfamily. A cDNA, c5126 (1737 bp), for P-450g was isolated from a lambda gt11 library synthesized from (+g) male rat liver mRNA. Sequence analysis of the clone, c5126, revealed an open reading frame of 1473 nucleotides, which encodes for a 490 amino acid polypeptide possessing the 30 NH2-terminal residues reported for cytochrome P-450 (M-3) (P-450g) [Matsumoto et al. (1986) J. Biochem. 100, 1359-1371]. A high degree of sequence similarity (greater than 70%) exists between c5126 and the published sequences of cDNAs for members of the IIC subfamily, while its sequence similarity to other subfamilies (IA, IIB, and IIIA) was much lower (less than 55%). RNA blot analysis utilizing an oligonucleotide probe specific for P-450g revealed that P-450g mRNA was expressed in livers of male but not female Sprague-Dawley (CD) and ACI rats, indicating that the sex difference was regulated pretranslationally. Furthermore, expression of P-450g mRNA was age dependent in livers of male ACI rats (a homozygous, phenotypically high P-450g strain). However, the mRNA for P-450g was expressed equally in livers of outbred male CD rats representing either the high (+g) or the low (-g) phenotype and of inbred ACI rats (+g) representing the high phenotype, indicating that the defect in (-g) rats does not reflect differences in expression of P-450g mRNA.

Amino Acid Sequence↗
PubMed1991

Molecular characterization of erythrocyte glycophorin C variants.

Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. Immunochemical and serologic studies have identified a number of glycophorin C variants that include the Yus, Gerbich, and Webb phenotypes. We report here the molecular characterization of these variants. Amplification of glycophorin C mRNA from the Yus phenotype, using two oligonucleotide primers that span the coding domain, generated a 338-bp fragment compared with a 395-bp fragment generated by amplification of normal glycophorin C mRNA. Sequencing of the mutant 338-bp fragment identified a 57-bp deletion that corresponds to exon 2 of the glycophorin C gene. Similar analysis showed deletion of 84-bp exon 3 in the Gerbich phenotype. In contrast to the generation of shorter than normal DNA fragments from mRNA amplification in the Yus and Gerbich phenotypes, amplification of mRNA from the Webb phenotype generated a normal-sized fragment. Sequencing of this DNA fragment showed an A----G substitution at nucleotide 23 of the coding sequence, resulting in the substitution of asparagine by serine. This modification accounts for the altered glycosylation of glycophorin C seen in this phenotype. These results have enabled us to characterize glycophorin C variants in three different phenotypes that involve deletions of exons 2 and 3 of the glycophorin C gene, as well as a point mutation in exon 1 that results in altered glycosylation of this protein.

Amino Acid Sequence↗
PubMed1980

Tissue-specific expression of mouse-alpha-amylase genes: nucleotide sequence of isoenzyme mRNAs from pancreas and salivary gland.

We have determined the nucleotide sequence of two different mouse alpha-amylase mRNAs, one found in the pancreas and the other in the salivary gland. The 1577 and 1659 nucleotide mRNAs from pancreas and salivary gland, respectively are the major alpha-amylase species which accumulate in each tissue. Differences in mRNA length are primarily in the 5' noncoding regions. Comparable portions of the mRNAs are 89% homologous. The mRNA sequences predict alpha-amylase precursor proteins of 508 and 511 amino acid residues, accounting for nearly the entire coding capacity of the mRNAs; differences in protein length occur as a result of a nine nucleotide segment present within the translated portion of salivary gland, but not pancreas, mRNA. The lengths and amino acid compositions of the predicted proteins concur with those determined empirically by others. These proteins differ 12% in amino acid sequence, explaining previously observed differences in net charge and antigenic properties. Finally, translation of the salivary gland alpha-amylase mRNA is not initiated at the AUG codon nearest the 5' terminus since that codon is almost immediately followed by the termination triplet UAA. This observation may have implications for the mechanism of translation initiation in eucaroytes.

Amino Acid Sequence↗
PubMed2004

Cloning of a kallikrein pseudogene.

OBJECTIVES: Kallikreins are a group of serine proteases clustered together on a small region of chromosome 19q13.4. Recent reports suggest that kallikreins are differentially expressed in malignancy and have potential as cancer biomarkers. The human kallikrein gene locus has now been fully characterized and 15 functional kallikreins were identified. Although many kallikrein pseudogenes have already been characterized in rodents, none have been identified in humans. METHODS AND RESULTS: In the current study, we identified the first human kallikrein pseudogene named PsiKLK1 and mapped it between the KLK2 and KLK4 genes. This pseudogene shares a moderate degree of similarity with the adjacent functional kallikreins. It has a conserved histidine residue of the catalytic triad of serine proteases and its surrounding motif, but lacks the aspartate and serine residues. Positions of some cysteine residues are also conserved in the pseudogene. This pseudogene lacks intronic sequences and should thus be classified as a processed pseudogene. EST and PCR analyses indicate that this pseudogene may be transcriptionally active, because mRNA was detected in many tissues including the prostate, testis, pituitary, and adrenal glands, as well as in tissues of the female genital organs. DISCUSSION: The mRNA sequence of the gene is, however, defective and is not predicted to code for a protein. Highly conserved sequences were found in the flanking region of the pseudogene, thus supporting the view that it evolved by retrotransposition. We also identified another serine protease fragment that has only the conserved histidine residue. The functional significance of the pseudogene and the other fragment is yet to be identified.

Amino Acid Sequence↗
PubMed1989

Is there a relationship between DNA sequences encoding peptide ligands and their receptors?

It has been suggested that the coding for a ligand and its receptor may have originated in inverse complementary strands of the same DNA. This would imply a deficiency of stop codons in the complementary strand of the ligand message sequence. We have sought evidence of such deficiencies by an analysis of the usage of selected codons in 23 human neuropeptide and hormone mRNA sequences. We have also searched directly for similarities between substance K or substance P and the substance K receptor. Although bovine proopiomelanocortin has an open reading frame for the full extent of the inverse complement of the coding region, this seems to be a unique case. The data as a whole do not support the hypothesis.

Amino Acid Sequence↗
PubMed2003

Functional complementation between a novel mammalian polygenic transport complex and an evolutionarily ancient organic solute transporter, OSTalpha-OSTbeta.

These studies identify an organic solute transporter (OST) that is generated when two novel gene products are co-expressed, namely human OSTalpha and OSTbeta or mouse OSTalpha and OSTbeta. The results also demonstrate that the mammalian proteins are functionally complemented by evolutionarily divergent Ostalpha-Ostbeta proteins recently identified in the little skate, Raja erinacea, even though the latter exhibit only 25-41% predicted amino acid identity with the mammalian proteins. Human, mouse, and skate OSTalpha proteins are predicted to contain seven transmembrane helices, whereas the OSTbeta sequences are predicted to have a single transmembrane helix. Human OSTalpha-OSTbeta and mouse Ostalpha-Ostbeta cDNAs were cloned from liver mRNA, sequenced, expressed in Xenopus laevis oocytes, and tested for their ability to functionally complement the corresponding skate proteins by measuring transport of [3H]estrone 3-sulfate. None of the proteins elicited a transport signal when expressed individually in oocytes; however, all nine OSTalpha-OSTbeta combinations (i.e. OSTalpha-OSTbeta pairs from human, mouse, or skate) generated robust estrone 3-sulfate transport activity. Transport was sodium-independent, saturable, and inhibited by other steroids and anionic drugs. Human and mouse OSTalpha-OSTbeta also were able to mediate transport of taurocholate, digoxin, and prostaglandin E2 but not of estradiol 17beta-d-glucuronide or p-aminohippurate. OSTalpha and OSTbeta were able to reach the oocyte plasma membrane when expressed either individually or in pairs, indicating that co-expression is not required for proper membrane targeting. Interestingly, OSTalpha and OSTbeta mRNAs were highly expressed and widely distributed in human tissues, with the highest levels occurring in the testis, colon, liver, small intestine, kidney, ovary, and adrenal gland.

Amino Acid Sequence↗
PubMed1990

Gene expression in bovine seminal vesicles.

Seminal vesicle secretion contributes significantly to the proteins of bovine seminal plasma. The following proteins from bull seminal vesicle were isolated and characterized: major protein (PDC 109), the basic proteins BUSI II, RNAse BS1, protein P6 and seminal antimicrobial protein (SAP). Using antibodies against the proteins BUSI II, RNAse BS1, SAP and major protein, the seminal vesicle epithelium was identified as the source of the respective antigens. The biosynthesis of bovine seminal vesicle secretory proteins was studied by cell free translation of poly (A)-RNA from seminal vesicles and the respective mRNAs were characterized by cDNA cloning. Recombinant clones (103) of a cDNA library of bull seminal vesicle poly (A) + RNA were screened by colony hybridisation using radioactively labelled synthetic probes. The respective clone containing the longest cDNA insert was sequenced. In case of major protein the Mr of the 134 amino acid residue precursor polypeptide was 15,480 as deduced from direct mRNA sequencing. The precursor sequence of 25 amino acid residues has a hydrophobic character and very likely constitutes a signal peptide, directing the protein towards the secretory pathway. The deduced amino acid sequence contained no consensus sequence indicative of N-glycosylation.

Amino Acid Sequence↗
PubMed1988

Insulin regulates prolactin secretion and messenger ribonucleic acid levels in pituitary cells.

The regulation of PRL production in GH3 rat pituitary tumor cells and normal rat pituitary cells exposed to insulin was studied. Cells were treated with semisynthetic human insulin for up to 5 days. Insulin (0.7 nM) stimulated PRL production by 30%, and cortisol (100 nM) suppressed it by 80% in GH3 cells. Insulin (3.5 nM) partially blocked the suppression of PRL secretion induced by up to 100 nM cortisol. Equimolar doses of insulin-like growth factor I failed to consistently stimulate either basal PRL secretion into the medium or cortisol-suppressed PRL in GH3 cells. A 65 nM concentration of insulin-like growth factor I was required to stimulate basal PRL secretion in GH3 cells. Relative levels of PRL mRNA sequences in both GH3 cells and normal rat pituitary cells were stimulated by insulin. When cells were incubated with cortisol (10 nM), PRL mRNA levels were suppressed to 40% of control values. Simultaneous incubation of cells with insulin (3.5 nM) partially reversed the cortisol-induced suppression of PRL mRNA to 60% of control values. The results show that insulin antagonizes cortisol-induced suppression of PRL. Physiological doses of insulin stimulate PRL production and PRL mRNA levels, suggesting a direct effect of insulin on either PRL gene transcription or stabilization of PRL mRNA in these cells.

Animals↗
PubMed1992

Retroviral expression of recombinant p47phox protein by Epstein-Barr virus-transformed B lymphocytes from a patient with autosomal chronic granulomatous disease.

Patients with chronic granulomatous disease (CGD) have recurrent infections resulting from a failure of phagocytic cells to produce superoxide. One third of CGD patients have an autosomal gene defect resulting in absence of p47phox protein, a cytoplasmic component critical to superoxide production by phagocytic cells. cDNA encoding p47phox has been cloned and recombinant p47phox (rp47phox) restores superoxide-generating activity to a cell-free assay containing cell membranes and cytosol from p47phox-deficient CGD neutrophils. The goal of the present study was to determine the feasibility of retrovirus mediated expression of rp47phox in the HL60 and U937 human hematopoietic cell lines, and in an Epstein-Barr virus transformed B-lymphocyte cell line (EBV-BCL) derived from a p47phox-deficient CGD patient. Normal EBV-BCL contain p47phox and generate small amounts of superoxide, while this CGD EBV-BCL lacks any detectable p47phox protein. Defective amphotropic retrovirus containing p47phox sequence inserted in the LXSN vector in sense and antisense orientations were used to transduce HL60, U937, and CGD EBV-BCL. p47phox mRNA sequence was detected in cells transduced with either sense or antisense retroviral constructs while rp47phox protein was detected only with the sense construct. The amount of rp47phox protein produced within these cells was greater than the native p47phox present in uninduced HL60 or U937 cells, but substantially less than that present in normal neutrophils, induced HL60 cells, or even normal EBV-BCL. Differentiation of transduced HL60 cells and the associated production of native p47phox in response to dimethyl sulfoxide was not affected. These studies demonstrate that retrovirus constructs can be used to mediate stable expression of rp47phox protein in human hematopoietic cell lines and can restore rp47phox protein within the cytosol of p47phox-deficient EBV-BCL from patients with CGD.

B-Lymphocytes↗
PubMed1988

Detection of messenger RNAs within single hemopoietic cells by in situ hybridization on small slide areas.

In situ hybridization provides a powerful tool to detect specific mRNA sequences at the cellular level. We have applied a modified in situ hybridization technique using specifically prepared regular glass microscope slides to evaluate mRNA levels in cells of small samples. Cells were derived from in vitro colonies or isolated by fluorescence-activated cell sorting and deposited on the slides. These slides were coated with polysiloxane, sparing small circular areas where adherent cells attach and can be grown directly; after preincubation of the collection areas with fibronectin, the slides can also be used to deposit and to grow nonadherent cells. In situ hybridization was performed with 35S-labeled probes. Acetylation of the slides and the cells prior to hybridization, the addition of vanadyl-ribonucleoside complexes, and a prehybridization step were found to be necessary to optimize signal-to-noise ratios, as shown by evaluation of c-myc-specific mRNA in phytohemagglutinin-stimulated T4-lymphocytes. This technique might be very useful to study mRNA expression in small samples of hemopoietic cells.

Cell Separation↗
PubMed1985

Coordinate expression of ribosomal protein mRNAs following auxin treatment of soybean hypocotyls.

Nine recombinant plasmids containing soybean (Glycine max. var. Wayne) ribosomal protein mRNA sequences were identified by hybrid selection-translation followed by gel electrophoresis. Individual plasmids were used to determine the amount of ribosomal protein mRNA in soybean hypocotyls at various times after application of the synthetic auxin (2,4-dichlorophenoxy)acetic acid. Results from these experiments indicate that the relative abundance of ribosomal protein mRNAs increases 3-8-fold within 24 hr of (2,4-dichlorophenoxy)acetic acid application. Earlier in vitro translation data (Gantt, J. S., and Key, J. L. (1983) Biochemistry 22, 4131-4139) also indicated that the ribosomal protein message levels increase following treatment with auxin. However, the two methods of quantifying mRNA lead to significantly different values for the amount of induction for three ribosomal protein mRNAs. The cause of these differences is not known. An increase in the level of the ribosomal protein mRNAs can be detected 1 h after treatment with (2,4-dichlorophenoxy)acetic acid. The similar kinetics of induction of these messages suggests that their levels are coordinately regulated.

Cloning, Molecular↗