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PubMed2001

Epidermal growth factor receptor signaling and the invasive phenotype of ovarian carcinoma cells.

BACKGROUND: Most (70%-100%) ovarian carcinomas express high levels of the epidermal growth factor receptor (EGFR). To examine the relationship between EGFR and the invasive phenotype, we assessed integrin expression, adhesion, matrix metalloproteinase (MMP) activity, and migration in ovarian cancer cells in which EGFR expression was modified. METHODS: NIH:OVCAR-8 human ovarian carcinoma cells were transfected with an expression vector containing the human EGFR complementary DNA in an antisense orientation (EGFR-antisense cells) or the vector alone (vector control cells). We compared vector control and EGFR-antisense cells for cell morphology and adhesion by light microscopy, expression of alpha(6)- and alpha(3)-integrin subunits by flow cytometry, MMP and tissue inhibitor of MMP (TIMP) activity by zymography, and migration by a wound migration assay. In some experiments, EGFR kinase activity in parental cells was inhibited by treatment with PD153035. All statistical tests were two-sided. RESULTS: EGFR-antisense cells were morphologically distinct from vector control cells and had a selective decrease in adhesion to laminin-1 that was not observed with vector control cells (P = .008) or on other extracellular matrix substrates. Compared with vector control cells, cell surface alpha(6)-integrin expression decreased by approximately 80% (difference = 78.7%; 95% confidence interval [CI] = 77.8% to 79.6), MMP-9 activity decreased by approximately 50%, and TIMP activity increased by approximately 50% in EGFR-antisense cells. Vector control cells were highly motile (5.51 arbitrary distance unit; 95% CI = 4.98 to 6.04), whereas the EGFR-antisense cells were not (0.99 arbitrary distance units; 95% CI = 0.38 to 1.60). The morphology and integrin profile of NIH:OVCAR-8 parental cells treated with PD153035 were similar to those of the EGFR-antisense cells. CONCLUSIONS: Reduced EGFR expression in ovarian carcinoma cells decreased their adhesion to laminin-1, expression of the alpha(6)-integrin subunit (a well-characterized laminin-1 receptor), and MMP-9 activity. These data support the hypothesis that EGFR overexpression in ovarian cancer cells results in multiple phenotypic changes that enhance the invasive phenotype.

Antigens, CD↗

PubMed2001

Prostasin, a potential serum marker for ovarian cancer: identification through microarray technology.

BACKGROUND: Screening biomarkers for ovarian cancer are needed because of its late stage at diagnosis and poor survival. We used microarray technology to identify overexpressed genes for secretory proteins as potential serum biomarkers and selected prostasin, a serine protease normally secreted by the prostate gland, for further study. METHODS: RNA was isolated and pooled from three ovarian cancer cell lines and from three normal human ovarian surface epithelial (HOSE) cell lines. Complementary DNA generated from these pools was hybridized to a microarray slide, and genes overexpressed in the cancer cells were identified. Real-time quantitative polymerase chain reaction was used to examine prostasin gene expression in ovarian cancer and HOSE cell lines. Anti-prostasin antibodies were used to examine prostasin expression and to measure serum prostasin by an enzyme-linked immunosorbent assay in 64 case patients with ovarian cancer and in 137 control subjects. Previously determined levels of CA 125, an ovarian cancer marker, were available from about 70% of all subjects. All statistical tests were two-sided. RESULTS: Prostasin was detected by immunostaining more strongly in cancerous ovarian epithelial cells and stroma than in normal ovarian tissue. The mean level of serum prostasin was 13.7 microg/mL (95% confidence interval [CI] = 10.5 to 16.9 microg/mL) in 64 case patients with ovarian cancer and 7.5 microg/mL (95% CI = 6.6 to 8.3 microg/mL) in 137 control subjects (P<.001, after adjustment for the subject's age, year of collection, and specimen quality). In 14 of 16 case patients with both preoperative and postoperative serum samples, postoperative prostasin levels were statistically significantly lower than preoperative levels (P =.004). In 37 case patients with nonmucinous ovarian cancer and in 100 control subjects for whom levels of CA 125 and prostasin were available, the combination of markers gave a sensitivity of 92% (95% CI = 78.1% to 98.3%) and a specificity of 94% (95% CI = 87.4% to 97.7%) for detecting ovarian cancer. CONCLUSIONS: Prostasin is overexpressed in epithelial ovarian cancer and should be investigated further as a screening or tumor marker, alone and in combination with CA 125.

Adult↗

PubMed2002

Coexpression of glucose transporter 1 and matrix metalloproteinase-2 in human cancers.

BACKGROUND: Cancer cells express higher levels of glucose transporter proteins (Gluts) than do normal cells. Glut-1 overexpression is associated with invasiveness. Because matrix metalloproteinase-2 (MMP-2) is also overexpressed in cancer cells and is associated with invasiveness, we tested the hypothesis that Glut-1 may regulate MMP-2 expression. METHODS: We transiently transfected Glut-1 complementary DNA (cDNA) or Glut-1 antisense oligonucleotides in the human rhabdomyosarcoma cell line RD and analyzed MMP-2 mRNA expression and cell invasiveness. Empty vector and sense oligonucleotides were used for controls. We analyzed MMP-2 promoter activity in transfectants with a luciferase reporter assay and with p53 and Ets-1 gel mobility shift assays. Eight human cancer cell lines and 80 human cancer specimens were analyzed for coexpression of Glut-1 and MMP-2 by western blot and immunohistochemical analyses, respectively. RESULTS: Overexpression of Glut-1 in RD cells increased MMP-2 expression 4.3-fold (95% confidence interval [CI] = 3.7-fold to 4.9-fold) and invasiveness 3.2-fold (95% CI = 2.6-fold to 3.8-fold) relative to control transfected cells. Conversely, suppression of Glut-1 expression by antisense oligonucleotides decreased MMP-2 expression by 71.5% (95% CI = 71.1% to 71.9%) and invasiveness by 53.0% (95% CI = 47.5% to 58.5%). Glut-1-mediated MMP-2 expression involved the binding of the transcription factor p53 but not Ets-1. All eight human cancer cell lines coexpressed Glut-1 and MMP-2 by western blotting, and 45 of 80 human tumor tissues coexpressed Glut-1 and MMP-2 by immunohistochemistry. CONCLUSIONS: MMP-2 expression and cell invasiveness are tightly associated with Glut-1 expression in human cancer cell lines. Because suppression of Glut-1 decreased MMP-2 expression and cancer cell invasion, Glut-1 could be a target for therapy of various cancers that overexpress Glut-1.

Blotting, Northern↗

PubMed2005

Spliced leader RNA-mediated trans-splicing in phylum Rotifera.

In kinetoplastids, Euglena, and four metazoan phyla, trans-splicing has been described as a mechanism for the generation of mature messenger RNAs (mRNAs): 5'-ends of precursor mRNAs are replaced by a short spliced leader (SL) exon from a small SL RNA. Although the full phylogenetic range is unknown, trans-splicing has not been found in vertebrates, insects, plants, or yeast. In animal groups where it does occur, i.e., nematodes, cnidarians, platyhelminths, and primitive chordates, SL RNAs do not show sequence relatedness across phyla. The apparently sporadic phylogenetic distribution and the lack of SL RNA homology have led to opposing hypotheses on its evolution, involving either an ancient origin followed by loss in multiple lineages or independent acquisition in several taxa. Here we present evidence for the occurrence of trans-splicing in bdelloid rotifers (Bdelloidea, Rotifera). A common 23-nt sequence, representing the SL exon-diagnostic of SL RNA-mediated trans-splicing-was found at the 5'-end of at least 50%-65% of mRNAs from Adineta ricciae and Philodina sp. The trans-splicing pattern in bdelloid rotifers can be unusually complex, as observed in transcripts from a heat shock protein gene, hsp82-1, where the SL exon was spliced to three alternative positions. Bdelloid rotifer SL RNAs were found to be 105 or 106 nt long and comprised the SL sequence, a conserved splice donor site and an intron containing a putative spliceosome-binding motif. Intriguingly, some similarity of rotifer SL RNA sequence and predicted secondary structure was seen to that of the predominant SL1 RNA of nematodes, although it is unlikely that this demonstrates homology. In addition, sequence corresponding to the rotifer SL exon was found at the 5'-end of a number of full-length complementary DNA (cDNA) clones in a rice (Oryza sativa) database. None of these cDNAs gave a close match with homologous plant genes, suggesting that a small but significant portion of the rice expressed sequence tag database represents sequences derived from rotifers. In summary, the description of SL-mediated trans-splicing in Rotifera extends its representation to at least five metazoan phyla, making it increasingly probable that this is a phylogenetically widespread and therefore ancient phenomenon.

Animals↗

PubMed2005

Evolutionary history of 4.5SH RNA.

4.5SH RNA is a 94-nt small RNA with unknown function. This RNA is known to be present in the mouse, rat, and hamster cells; however, it is not found in human, rabbit, and chicken. In the mouse genome, the 4.5SH RNA gene is a part of a long (4.2 kb) tandem repeat ( approximately 800 copies) unit. Here, we found that 4.5SH RNA genes are present only in rodents of six families that comprise the Myodonta clade: Muridae, Cricetidae, Spalacidae, Rhizomyidae, Zapodidae, and Dipodidae. The analysis of complementary DNA derived from the rodents of these families showed general evolutionary conservation of 4.5SH RNA and some intraspecific heterogeneity of these RNA molecules. 4.5SH RNA genes in the Norway rat, mole rat, hamster and jerboa genomes are included in the repeated sequences. In the jerboa genome these repeats are 4.0-kb long and arranged tandemly, similar to the corresponding arrangements in the mouse and rat genomic DNA. Sequencing of the rat and jerboa DNA repeats containing 4.5SH RNA genes showed fast evolution of the gene-flanking sequences. The repeat sequences of the distantly related rodents (mouse and rat vs. jerboa) have no apparent similarity except for the 4.5SH RNA gene itself. Conservation of the 4.5SH RNA gene nucleotide sequence indicates that this RNA is likely to be under selection pressure and, thus, may have a function. The repeats from the different rodents have similar lengths and contain many simple short repeats. The data obtained suggest that long insertions, deletions, and simple sequence amplifications significantly contribute in the evolution of the repeats containing 4.5SH RNA genes. The 4.5SH RNA gene seems to have originated 50-85 MYA in a Myodonta ancestor from a copy of the B1 short interspersed element. The amplification of the gene with the flanking sequences could result from the supposed cellular requirement of the intensive synthesis of 4.5SH RNA. Further Myodonta evolution led to dramatic changes of the repeat sequences in every lineage with the conservation of the 4.5SH RNA genes only. This gene, like some other relatively recently originated genes, could be a useful model for studying generation and evolution of non-protein-coding genes.

Animals↗

PubMed1999

Co-expression of integrin-associated protein (IAP/CD47) and its ligand thrombospondin-1 on human granulosa and large luteal cells.

In the present study, we have raised a monoclonal antibody (mAb) designated OG-4 against human granulosa cells (GC). By immunohistochemistry, the expression of OG-4 antigen was observed on human GC and large luteal cells, but not on thecal and small luteal cells. A complementary DNA (cDNA) clone encoding OG-4 antigen was screened and isolated by a panning method using OG-4 mAb from a human corpus luteum (CL) cDNA library that was expressed transiently in COS-7 cells. Nucleotide sequencing revealed that OG-4 antigen was identical to integrin-associated protein (IAP)/CD47 antigen. Subsequent reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the isoform 2 mRNA of IAP is predominantly expressed in human GC and CL. The expression of thrombospondin-1 (TSP-1), which is a ligand for IAP, was also observed in human GC by immunocytochemistry and RT-PCR. Co-expression of IAP and TSP-1 on human GC may suggest that TSP-1 has a physiological role in GC function possibly via IAP in an autocrine fashion.

Adult↗

PubMed1995

Cloning of cDNAs encoding the 160 kDa subunit of the bovine cleavage and polyadenylation specificity factor.

3'-processing of mRNA precursors depends on several protein factors. One of them, cleavage and polyadenylation specificity factor (CPSF) is required for the cleavage of the mRNA precursor or as well as for the tail elongation reaction. We have obtained complementary DNA encoding the 160 kDa subunit, which had previously been shown to interact with the AAUAAA polyadenylation signal. The cDNAs code for an open reading frame of 1444 amino acids. The translated protein has a calculated molecular weight of 161 kDa and a predicted pl of 6.2. Polyclonal antibodies raised against a bacterially expressed fragment of the cDNA recognise 160 kDa subunit of purified calf thymus CPSF. The sequence contains a possible nuclear localisation signal but none of the known RNA binding motifs. It does, however, show sequence similarities to a UV-damaged DNA binding protein (UVdDb).

Amino Acid Sequence↗

PubMed1998

NMR solution structures of [d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2] and its unmodified control.

We present the high-resolution solution structures of a self-complementary DNA decamer duplex featuring a single alpha-anomeric nucleotide per strand encompassed by a set of 3'-3' and 5'-5' phosphodiester linkages, d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2, alphaT, and its unmodified control, d(GCGAATTCGC)2, obtained by restrained molecular dynamics. Interproton distance and deoxyribose ring torsion angle restraints were deduced from homonuclear NOESY and DQF-COSY data, respectively. For both the control and alphaT duplexes, excellent global convergence was observed from two different (A- and B-) starting models. The final average structures of the two duplexes are highly homologous, and overall possess the traits characteristic of right-handed B-DNA duplexes. However, localized differences between the two structures stem from the enhanced conformational exchange in the deoxyribose ring of the cytidine following the 5'-5' linkage, the C3'- exo pseudorotation phase angle of the alpha-nucleotide, and unusual backbone torsions in the 3'-3' and 5'-5' phosphodiester linkages. The structural data reported here are relevant to the design of antisense therapeutics comprised of these modifications.

DNA Probes↗

PubMed2003

Cc RNase: the Ceratitis capitata ortholog of a novel highly conserved protein family in metazoans.

Complementary DNA encoding a protein, designated Cc RNase, was isolated from the insect Ceratitis capitata. Deduced amino acid sequence analysis demonstrates that the Cc RNase has strong sequence homology with other uncharacterized proteins predicted from EST sequences belonging to different animal species, therefore defining a new protein family, which is conserved from Caenorhabditis elegans to humans. Phylogenetic analysis data in addition to extensive homolog searches in all available complete genomes suggested that all family members are true orthologs. Proteins belonging to this family are composed of 95-101 amino acids. The C.capitata orthologous protein was expressed in Escherichia coli. Despite the fact that the amino acid sequence of Cc RNase does not share any significant similarities with other known ribonucleases, our data give strong evidence in support of the assignment of enzymatic activity to the recombinant protein. The expressed molecule exhibits ribonucleolytic activity against poly(C) and poly(U) synthetic substrates, as well as rRNA. It is also demonstrated that expression of Cc RNase in E.coli inhibits growth of the host cells.

Amino Acid Sequence↗

PubMed2002

Improved quantitative real-time RT-PCR for expression profiling of individual cells.

The real-time quantitative polymerase chain reaction (rtqPCR) has overcome the limitations of conventional, time-consuming quantitative PCR strategies and is maturing into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). Expression profiling with single-cell resolution is highly desirable, in particular for complex tissues like the brain that contain a large variety of different cell types in close proximity. The patch-clamp technique allows selective harvesting of single-cell cytoplasm after recording of cellular activity. However, components of the cDNA reaction, in particular the reverse transcriptase itself, significantly inhibit subsequent rtqPCR amplification. Using undiluted single-cell cDNA reaction mix directly as template for rtqPCR, I observed that the amplification kinetics of rtqPCRs were dramatically altered in a non-systematic fashion. Here, I describe a simple and robust precipitation protocol suitable for purification of single-cell cDNA that completely removes inhibitory RT components without detectable loss of cDNA. This improved single-cell real-time RT-PCR protocol provides a powerful tool to quantify differential gene expression of individual cells and thus could complement global microarray-based expression profiling strategies.

Animals↗

PubMed1997

Molecular cloning and sequencing of cDNA encoding plasma countertrypin, a member of mammalian fetuin family, from the Mongolian gerbil, Meriones unguiculatus.

Complementary DNA clones coding for countertrypin were isolated from a liver cDNA library of the Mongolian gerbil, and sequenced. They contained one open reading frame encoding 348 amino acid residues, which were assigned to consist of an 18-residue signal peptide and a 330-residue mature protein. The amino acid sequence was about 74% identical with mouse countertrypin and rat fetuin, 60% with bovine fetuin, and 55% with human alpha2HS glycoprotein, indicating that this protein belongs to the mammalian fetuin family. The members of this family are known to consist of three domains, i.e., two tandemly arranged cystatin domains (D1 and D2) and an unrelated domain (D3) located at the C-terminal region. When compared with the other members of this family, D3, especially its N-terminal half, varies greatly with deletion or insertion as well as nucleotide substitutions even among three rodent species, i.e., gerbil, rat, and mouse. The sequence comparison also suggests that the conformation of human alpha2HS glycoprotein differs greatly from that of other members of this family. A molecular phylogenetic tree of 7 members, constructed on the basis of the synonymous substitution rate of D1 and D2, shows that the gerbil gene diverged prior to the separation of mouse and rat.

Amino Acid Sequence↗

PubMed1993

Thymidine phosphorylase activity associated with platelet-derived endothelial cell growth factor.

Partial complementary DNA (cDNA) for thymidine phosphorylase (dThdPase) was cloned by means of a polymerase chain reaction. There was complete sequence identity between the amino acid sequence deduced from the nucleotide sequence of a clone (288 nucleotides) and the residues of platelet-derived endothelial cell growth factor (PD-ECGF). The amino acid sequence of all four peptide fragments from purified human dThdPase could be aligned with that of PD-ECGF. Our data indicate that residues 125-244 of PD-ECGF are identical to the sequence of human dThdPase. The molecular weights of human dThdPase and recombinant PD-ECGF (rPD-ECGF) that lacks 10 amino acids at the amino terminal were 55 and 52 kDa, respectively. Anti-PD-ECGF antibody recognized dThdPase, and anti-dThdPase antibody recognized rPD-ECGF. rPD-ECGF had dThdPase activity and its specific activity was similar to that of purified human dThdPase. dThdPase activity and molecules were detected in COS cells transfected with human PD-ECGF cDNA, but not in nontransfected cells. The sizes of PD-ECGF and dThdPase in the transfected COS cells were identical. These data suggest that human dThdPase is identical to PD-ECGF.

Amino Acid Sequence↗

PubMed1998

Complex history of a chromosomal paralogy region: insights from amphioxus aromatic amino acid hydroxylase genes and insulin-related genes.

Aromatic amino acid hydroxylase (AAAH) genes and insulin-like genes form part of an extensive paralogy region shared by human chromosomes 11 and 12, thought to have arisen by tetraploidy in early vertebrate evolution. Cloning of a complementary DNA (cDNA) for an amphioxus (Branchiostoma floridae) hydroxylase gene (AmphiPAH) allowed us to investigate the ancestry of the human chromosome 11/12 paralogy region. Molecular phylogenetic evidence reveals that AmphiPAH is orthologous to vertebrate phenylalanine (PAH) genes; the implication is that all three vertebrate AAAH genes arose early in metazoan evolution, predating vertebrates. In contrast, our phylogenetic analysis of amphioxus and vertebrate insulin-related gene sequences is consistent with duplication of these genes during early chordate ancestry. The conclusion is that two tightly linked gene families on human chromosomes 11 and 12 were not duplicated coincidentally. We rationalize this paradox by invoking gene loss in the AAAH gene family and conclude that paralogous genes shared by paralogous chromosomes need not have identical evolutionary histories.

Amino Acid Sequence↗

PubMed1994

Comparative analysis of the zeta-crystallin/quinone reductase gene in guinea pig and mouse.

zeta-Crystallin is a novel nicotinamide adenine dinucleotide phosphate:quinone reductase, present at enzymatic levels in various tissues of different species, which is highly expressed in the lens of some hystricomorph rodents and camelids. We report here the complementary DNA (cDNA) cloning of zeta-crystallin from liver libraries in guinea pig (Cavia porcellus), where zeta-crystallin is highly expressed in the lens, and in the laboratory mouse (Mus musculus), where expression in the lens occurs only at enzymatic levels. A 5' untranslated sequence different from the one previously reported for the guinea pig lens cDNA was found in these clones. We also report the isolation of genomic clones including the complete guinea pig zeta-crystallin gene and the 5' region of this gene in mouse. These results show the presence of two promoters in the guinea pig zeta-crystallin gene, one responsible for expression at enzymatic levels and the other responsible for the high expression in the lens. The guinea pig lens promoter is not present in the mouse gene. This is the first example in which the recruitment of an enzyme as a lens crystallin can be explained by the acquisition of an alternative lens-specific promoter.

Amino Acid Sequence↗

PubMed2000

Effects of the oxidant potassium permanganate on the expression of gill metallothionein mRNA and its relationship to sublethal whole animal endpoints in channel catfish.

Potassium permanganate is an oxidant heavily used in fish culture. The effects of this compound were examined utilizing molecular (Metallothionein) and whole animal endpoints following an 8-week exposure to nominal concentrations of 0.5 (daily) and 1.0 and 2.0 mg/L (on alternate days) of potassium permanganate (PM). In order to measure MT, a complementary DNA clone of metallothionein (MT) was cloned and sequenced from the liver of channel catfish treated with a single injection of cadmium chloride (10 mg/kg). The cDNA was obtained by reverse transcriptase polymerase chain reaction (RT-PCR), using 3' rapid amplification of cDNA ends (RACE) technique. No significant correlation was observed with gill MT expression or sublethal endpoints indicative of toxicity (weight, length, condition index [CI], or liver somatic index [LSI). MT mRNA expression in gill was significantly reduced only after 8 weeks in the 2.0 mg/L treatment. Decreases in CI were observed in males at all time points after 4 weeks, at the 2.0 mg/L treatment concentration, with a NOEC of 1 mg/L. Reductions in LSI that were not dose dependent were also observed in both males and females throughout the 8-week study and no consistent reduction in weight gain or length was observed. These data demonstrate that minimal changes in sublethal effects occur in fish following 0.5-2.0 mg/L PM treatment after 4 weeks, but recovery from adverse effects is observed by 8 weeks, suggesting that acute (typically less than 1 week) treatment of channel catfish with PM would not significantly affect fish health.

Animals↗

PubMed2006

Development and validation of endogenous reference genes for expression profiling of medaka (Oryzias latipes) exposed to endocrine disrupting chemicals by quantitative real-time RT-PCR.

The quantitative real-time reverse transcription polymerase chain reaction (Q-RT-PCR) technique has been increasingly used in endocrine disrupting chemicals (EDCs) research. Usually, an appropriate endogenous control gene is critical for Q-RT-PCR to normalize the errors and sample-to-sample variations that occur in the course of tissue collection, RNA isolation, and RT-PCR. In this study, we cloned ribosomal protein L7 (RPL-7) from medaka (Oryzias latipes), and then used Q-RT-PCR to study its transcription characteristics and those of glyceraldehyde-3-phosphate dehydrogenase, beta-actin, mitochondrial 16S ribosomal RNA (16S rRNA), and 18S rRNA. Of the five genes, RPL-7 and 18S rRNA were expressed with the less variance among the same tissue samples, in different tissues, and stages of development and were unaffected by EDCs exposure. The expression levels of RPL-7 among different tissues were between 9.76 x 10(6) +/- 9.49 x 10(5) and 1.39 x 10(7) +/- 1.69 x 10(6) copies/microg RNA but those of 18S rRNA were as high as 4.48 x 10(11) +/- 5.95 x 10(10) to 5.90 x 10(11) +/- 1.21 x 10(10) copies/microg RNA, which is above the usual detection scope of Q-RT-PCR if no complementary DNA reaction dilution is performed. As a result, RPL-7 is the single suitable endogenous control gene for expression profiling in future studies, especially in studies on the EDCs issue using medaka.

Amino Acid Sequence↗

PubMedDate not supplied

Interstrain pseudorecombinants of cowpea chlorotic mottle virus: effects on systemic spread and symptom formation in soybean and cowpea.

Full-length complementary DNA (cDNA) copies of genomic RNA1, RNA2, and RNA3 segments of cowpea chlorotic mottle virus (CCMV) strains D, N, and S were synthesized using polymerase chain reaction and were cloned downstream of a T7 RNA polymerase promoter. Mixtures of the homologous in vitro-transcribed RNAs produced typical CCMV symptoms when inoculated on soybean (cv. Bragg) and cowpea (cv. California Blackeye) plants. Using either gel-purified or in vitro-transcribed CCMV RNA components, the pseudorecombinants SSD, SSN, DDS, and NNS were constructed. The properties of these pseudorecombinants were tested by comparing the induced symptoms, virus concentration, and systemic spread. These studies revealed a direct involvement of RNA3 genetic information in necrotic lesion formation and in breakage of CCMV resistance in soybean. Exchanges between RNA1 or RNA2 components further affected the biological features of reassorted CCMV variants. The true nature of the pseudorecombinants was confirmed by reconstitution of the parental strains from the corresponding pseudorecombinant RNA components.

Base Sequence↗

PubMed2003

Gene and protein expression in the epididymis of infertile c-ros receptor tyrosine kinase-deficient mice.

Transgenic male mice bearing inactive mutations of the receptor tyrosine kinase c-ros lack the initial segment of the epididymis and are infertile. Several techniques were applied to determine differences in gene expression in the epididymal caput of heterozygous fertile (HET) and infertile homozygous knockout (KO) males that may explain the infertility. Complementary DNA arrays, gene chips, Northern and Western blots, and immunohistochemistry indicated that some proteins were downregulated, including the initial segment/proximal caput-specific genes c-ros, cystatin-related epididymal-spermatogenic (CRES), and lipocalin mouse epididymal protein 17 (MEP17), whereas other caput-enriched genes (glutathione peroxidase 5, a disintegrin and metalloproteinase [ADAM7], bone morphogenetic proteins 7 and 8a, A-raf, CCAAT/enhancer binding protein beta, PEA3) were unchanged. Genes normally absent from the initial segment (gamma-glutamyltranspeptidase, prostaglandin D2 synthetase, alkaline phosphatase) were expressed in the undifferentiated proximal caput of the KO. More distally, lipocalin 2 (24p3), CRISP1 (formerly MEP7), PEBP (MEP9), and mE-RABP (MEP10) were unchanged in expression. Immunohistochemistry and Western blots confirmed the absence of CRES in epididymal tissue and fluid and the continued presence of CRES in spermatozoa of the KO mouse. The glutamate transporters EAAC1 (EAAT3) and EAAT5 were downregulated and upregulated, respectively. The genes of over 70 transporters, channels, and pores were detected in the caput epididymidis, but in the KO, only three were downregulated and six upregulated. The changes in these genes could affect sperm function by modifying the composition of epididymal fluid and explain the infertility of the KO males. These genes may be targets for a posttesticular contraceptive.

Animals↗