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The sequences of two cloned rat pancreatic amylase cDNAs comprising 95% of the mRNA sequence are reported. Analysis of cloned rat genomic DNA fragments using cloned cDNA probes indicates that the rat genome contains multiple closely related amylase genes in which the cDNA sequences are distributed within a region 9 kilobases in length and are interrupted by at least seven intervening sequences.
The mosses and flowering plants diverged >400 million years ago. The mosses have haploid-dominant life cycles, whereas the flowering plants are diploid-dominant. The common ancestors of land plants have been inferred to be haploid-dominant, suggesting that genes used in the diploid body of flowering plants were recruited from the genes used in the haploid body of the ancestors during the evolution of land plants. To assess this evolutionary hypothesis, we constructed an EST library of the moss Physcomitrella patens, and compared the moss transcriptome to the genome of Arabidopsis thaliana. We constructed full-length enriched cDNA libraries from auxin-treated, cytokinin-treated, and untreated gametophytes of P. patens, and sequenced both ends of >40,000 clones. These data, together with the mRNA sequences in the public databases, were assembled into 15,883 putative transcripts. Sequence comparisons of A. thaliana and P. patens showed that at least 66% of the A. thaliana genes had homologues in P. patens. Comparison of the P. patens putative transcripts with all known proteins, revealed 9,907 putative transcripts with high levels of similarity to vascular plant genes, and 850 putative transcripts with high levels of similarity to other organisms. The haploid transcriptome of P. patens appears to be quite similar to the A. thaliana genome, supporting the evolutionary hypothesis. Our study also revealed that a number of genes are moss specific and were lost in the flowering plant lineage.
The sequences of seven new Trypanosoma brucei kinetoplast DNA minicircles were obtained. A detailed comparative analysis of these sequences and those of the 18 complete kDNA minicircle sequences from T. brucei available in the database was performed. These 25 different minicircles contain 86 putative gRNA genes. The number of gRNA genes per minicircle varies from 2 to 5. In most cases, the genes are located between short imperfect inverted repeats, but in several minicircles there are inverted repeat cassettes that did not contain identifiable gRNA genes. Five minicircles contain single gRNA genes not surrounded by identifiable repeats. Two pairs of closely related minicircles may have recently evolved from common ancestors: KTMH1 and KTMH3 contained the same gRNA genes in the same order, whereas KTCSGRA and KTCSGRB contained two gRNA genes in the same order and one gRNA gene specific to each. All minicircles could be classified into two classes on the basis of a short substitution within the highly conserved region, but the minicircles in these two classes did not appear to differ in terms of gRNA content or gene organization. A number of redundant gRNAs containing identical editing information but different sequences were present. The alignments of the predicted gRNAs with the edited mRNA sequences varied from a perfect alignment without gaps to alignments with multiple mismatches. Multiple gRNAs overlapped with upstream gRNAs, but in no case was a complete set of overlapping gRNAs covering an entire editing domain obtained. We estimate that a minimum set of approximately 65 additional gRNAs would be required for complete overlapping sets. This analysis should provide a basis for detailed studies of the evolution and role in RNA editing of kDNA minicircles in this species.
A three-dimensional collagen lattice can provide skin fibroblasts with a cell culture environment that simulates normal dermis. Such a collagen matrix environment regulates interstitial collagenase (type I metalloproteinase [MMP-1], collagenase-1) and collagen receptor alpha2 subunit mRNA expression in both unstimulated or platelet-derived growth factor-stimulated dermal fibroblasts (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239-249). Here we report that the collagen gel can signal protein kinase C (PKC)-zeta activation in human dermal fibroblasts. An in vitro kinase assay demonstrated that autophosphorylation of PKC-zeta immunoprecipitates was markedly increased by a collagen matrix. In contrast, no alteration in PKC-zeta protein levels or intracellular location was observed. DNA binding activity of nuclear factor kappaB (NF-kappaB), a downstream regulatory target of PKC-zeta, was also increased by fibroblasts grown in collagen gel. The composition of the NF-kappaB/Rel complexes that contained p50, was not changed. The potential role of PKC-zeta in collagen gel-induced mRNA expression of collagen receptor alpha2 subunit and human fibroblast MMP-1 was assessed by the following evidence. Increased levels of alpha2 and MMP-1 mRNA in collagen gel-stimulated fibroblasts were abrogated by bisindolylmaleimide GF 109203X and calphostin C, chemical inhibitors for PKC, but retained when cells were depleted of 12-myristate 13-acetate (PMA)-inducible PKC isoforms by 24 h of pretreatment with phorbol PMA. Antisense oligonucleotides complementary to the 5' end of PKC-zeta mRNA sequences significantly reduced the collagen lattice-stimulated alpha2 and MMP-1 mRNA levels. Taken together, these data indicate that PKC-zeta, a PKC isoform not inducible by PMA or diacylglycerol, is a component of collagen matrix stimulatory pathway for alpha2 and MMP-1 mRNA expression. Thus, a three-dimensional collagen lattice maintains the dermal fibroblast phenotype, in part, through the activation of PKC-zeta.
A new method is described for biotinylation of oligonucleotide probes for use in molecular hybridization reactions. Biotin-11-dUTP residues were added enzymatically, using terminal deoxynucleotidyl transferase, to the 3' terminus of a synthetic oligonucleotide prepared from the known nucleotide sequence for adenosine deaminase. The biotinylated probe was hybridized to DNA and mRNA selectively immobilized on nitrocellulose and detected by sequential incubation of the nitrocellulose membrane with avidin and biotinylated polyalkaline phosphatase, followed by colorimetric development. The biotinylated oligonucleotide probe proved useful for the qualitative detection of complementary DNA and mRNA sequences but was unsatisfactory for quantitative determinations using reflective densitometry.
A series of overlapping recombinant clones, which cover the vitellogenin gene, has been isolated from a phage-lambda linked chicken gene library. The DNA of the overlapping clones spans 28 kb of contiguous DNA sequences in the chicken genome. Electron microscopic analysis of hybrids between vitellogenin mRNA and the genomic clones indicates that the chicken vitellogenin gene has a length of approximately 22 kb, about 3.8 times the size of the mRNA. The mRNA sequence is interrupted by at least 33 intervening sequences (introns). Comparison with the vitellogenin gene A2 from Xenopus laevis (Wahli et al., 1980, Cell 20: 107-117) indicates conservation of the number and length of the exons during evolution. Heteroduplex analysis reveals a short stretch of sequence homology between the genes from chicken and frog.
A procedure is described for the chromatographic analysis of RNA under fully denaturing conditions on cross-linked Sepharose. Chromatography of DNA . RNA hybrids, poly(C) . poly(G) hybrids and complexes of poly(C) . hnRNA on Sepharose CL in pure formamide at 46 degrees C leads to denaturation and strand separation of the hybrid structures. Using this procedure, nuclear RNA from duck immature red blood cells was resolved according to molecular size and assayed for the presence of globin mRNA sequences by hybridization with complementary DNA. Two size classes of putative globin mRNA precursor molecules were detected at an elution position corresponding to 14--18 S and 23--28 S. As judged from chromatographic analysis on poly(U)-Sepharose, about 70% of the 14--18-S globin precursor RNA is polyadenylated while only 11% of the putative 23--28-S precursor RNA has a poly(A) tract. Inhibition of transcription by actinomycin D and pulse-chase experiments indicate a half-life of less than 7.5 min for these precursor RNA species.
The aim of the present work was to determine whether the accumulation of Ig mRNA in myeloma cells is due to a high rate of production or to a high stability of these molecules. Specific mRNAs for the light and heavy polypeptide chains of IgG were isolated from the murine MPC-11 myeloma tumor cells by immune precipitation of polysomes which synthesize these chains. It was found that the immune-precipitated polysomes were enriched 10--30-fold in the gamma and chi mRNA sequences respectively. In the wheat germ cell-free system the chi mRNA preparation was translated mainly into three polypeptides of Mr 25 000, 18 000, and 15 000. The method of immune precipitation of polysomes was also used to characterize three variant clones of MPC-11 myeloma. It was found that little if any gamma-chain polysomes are present in the L-chain producer and non-producer clones, while a substantial amount of chi-chain polysomes was present in the non-producer clone. This may be due to the presence in the non-producer cells of the constant region chi-chain fragment. In order to determine the relative synthesis rate of chi and gamma mRNAs, pulse-labelled polysomes were immune precipitated using antibodies to chi and gamma chains. It was found that chi and gamma mRNA molecules are produced at a very high relative rate each accounting for 10--15% of the total labeled mRNA after 1 h of labeling. These values are higher than the steady-state pool size of chi and gamma mRNA, which was 5--6%, and indicates that the half-life of these molecules is not unusually high. It is concluded that the amplified synthesis of immunoglobulin chains in myeloma cells is mainly due to a high rate of production of translatable chi and gamma mRNAs.
The genes encoding carbamoylphosphate synthetase from Pseudomonas aeruginosa PAO1 were cloned in Escherichia coli. Deletion and transposition analysis determined the locations of carA, encoding the small subunit, and carB, encoding the large subunit, on the chromosomal insert. The nucleotide sequence of carA and the flanking regions was determined. The derived amino acid sequence for the small subunit of carbamoylphosphate synthetase from P. aeruginosa exhibited 68% homology with its counterparts in E. coli and Salmonella typhimurium. The derived sequences in the three organisms were essentially identical in the three polypeptide segments that are conserved in glutamine amidotransferases but showed low homology at the amino- and carboxy-terminal regions. The amino-terminal amino acid sequences were determined for the large and small subunits. The first 15 amino acids of the large subunit were identical to those derived from the carB sequence. However, comparison of the derived sequence for carA with the amino-terminal amino acid sequence for the small subunit suggested that codons 5 to 8 are not translated. The DNA sequence for the region encompassing these four codons was confirmed by direct sequencing of chromosomal DNA after amplification by the polymerase chain reaction. The mRNA sequence was also deduced by in vitro synthesis of cDNA, enzymatic amplification, and sequencing, confirming that 12 nucleotides in the 5' terminal of carA are transcribed but are not translated.
KEY POINTS: A novel nonsense variant ( mcy ) in ankyrin repeat and sterile alpha motif domain-containing 6 ( Anks6 ) promotes rapid disease progression in the Han:SPRD- Cy rat carrying a missense variant in Anks6 . mcy-/- rats exhibit prenatal lethality characterized by laterality and cardiovascular abnormalities. These findings indicate that ANKS6 nonfunction in rats leads to prenatal lethality, whereas misfunction leads to polycystic kidney disease development. BACKGROUND: Polycystic kidney disease (PKD) encompasses a group of genetic disorders characterized by the proliferation of fluid-filled renal cysts, leading to progressive renal failure and death. A key feature of PKD is its variable expressivity across patients, even when caused by the same variant, highlighting the importance of genetic background in PKD expression. METHODS: We identified an ostensibly healthy Sprague Dawley rat line with a variant that modifies PKD expressivity in Han:SPRD- Cy rats (caused by a missense variant [p.Arg717Trp] in the ankyrin repeat and sterile alpha motif domain-containing 6 [ Anks6 ] gene), which we named mcy (modifier of Cy ). We used whole-genome sequencing and segregation analysis to identify the mcy variant, quantitative PCR and mRNA sequencing to evaluate its effects on gene expression, western blotting and immunohistochemistry to assess its protein consequences, and ultrasound and histology to examine its impact on rat embryonic development. RESULTS: We identified a nonsense variant in the Anks6 gene as the genetic basis of the mcy phenotype (c.1126G>T [p.Glu376X]). Although mcy+/- rats are ostensibly healthy and do not develop PKD, mcy-/- rats exhibit laterality defects and die prenatally at E16.5 because of apparent perturbations in cardiovascular development. Notably, mcy+/-Cy+/- rats develop PKD much more rapidly than Cy+/- rats, and in a timeframe consistent with Cy-/-rats . Transcripts with the mcy variant allele seem to undergo nonsense-mediated decay, and no ANKS6 protein is detected. However, gene expression patterns in the kidneys did not differ significantly between age-matched mcy+/+ and mcy+/- rats, indicating that ANKS6 insufficiency does not cause PKD. CONCLUSIONS: We identified a novel nonsense variant in Anks6 . The findings indicate that the absence of wild-type ANKS6 accelerates PKD development in the Han:SPRD- Cy rat and that complete ANKS6 deficiency prevents normal embryonic development in rats.
In 1980 the clinical syndrome of X-linked hypogammaglobulinemia and isolated growth hormone deficiency (XLA/GHD) was described. XLA/GHD patients have reduced serum levels of Ig and normal cell-mediated immunity, and thus resemble patients with Bruton's X-linked agammaglobulinemia (XLA). However, XLA/GHD patients also have isolated GHD. Mutations and deletions in the Bruton's tyrosine kinase gene (BTK) are responsible for Bruton's XLA. We investigated BTK gene expression in an XLA/GHD patient from the family originally described by Northern analysis, cDNA sequencing, and Western analysis of protein production using mAb to BTK. BTK mRNA was normal in size and abundance, and the mRNA sequence was normal over the coding region, except for a single silent mutation. BTK protein was present in normal amounts in PBMC of this patient. Thus, at the molecular level, XLA/GHD is a different disease entity from Bruton's XLA. These results suggest that undescribed genes critical for B cell development and growth hormone production exist on the X chromosome.
The objective of this study was to determine if mRNA sequences downstream of the translation initiation codon are important for translation of plastid mRNAs. We have employed a transgenic approach, measuring accumulation of the neomycin phosphotransferase (NPTII) reporter enzyme translationally fused with 14 N-terminal amino acids encoded in the rbcL or atpB plastid genes. NPTII accumulation from wild-type and mutant rbcL and atpB segments was compared. We report that silent mutations in the rbcL segment reduced NPTII accumulation 35-fold. In contrast, mutations in the atpB mRNA reduced NPTII accumulation only moderately from approximately 7% (w/w) to approximately 4% (w/w) of the total soluble cellular protein, indicating that the importance of sequences downstream of the translation initiation codon are dependent on the individual mRNA. Information provided here will facilitate transgene design for high-level expression of recombinant proteins in chloroplasts by translational fusion with the N-terminal segment of highly expressed plastid genes or by introduction of silent mutations in the N-terminal part of the coding region.
By means of immunological screening, a cDNA clone bearing the mRNA sequence for rat transferrin was isolated from a cDNA library of rat liver mRNA. The amounts of transferrin mRNA in livers of analbuminemic rats (NAR, Nagase analbuminemia rats) and normal rats were determined by RNA blot hybridization using a cloned transferrin cDNA probe. The level of transferrin mRNA in the NAR liver was about 1.7 times that in the normal rat liver. These findings suggest that the enhanced synthesis of transferrin in the NAR liver resulted from an increase in the transferrin mRNA level.
A simple membrane-strip-based biosensor assay has been combined with a nucleic acid sequence-based amplification (NASBA) reaction for rapid (4 h) detection of a small number (ten) of viable B. anthracis spores. The biosensor is based on identification of a unique mRNA sequence from one of the anthrax toxin genes, the protective antigen ( pag), encoded on the toxin plasmid, pXO1, and thus provides high specificity toward B. anthracis. Previously, the anthrax toxins activator ( atxA) mRNA had been used in our laboratory for the development of a biosensor for the detection of a single B. anthracis spore within 12 h. Changing the target sequence to the pag mRNA provided the ability to shorten the overall assay time significantly. The vaccine strain of B. anthracis (Sterne strain) was used in all experiments. A 500-microL sample containing as few as ten spores was mixed with 500 microL growth medium and incubated for 30 min for spore germination and mRNA production. Thus, only spores that are viable were detected. Subsequently, RNA was extracted from lysed cells, selectively amplified using NASBA, and rapidly identified by the biosensor. While the biosensor assay requires only 15 min assay time, the overall process takes 4 h for detection of ten viable B. anthracis spores, and is shortened significantly if more spores are present. The biosensor is based on an oligonucleotide sandwich-hybridization assay format. It uses a membrane flow-through system with an immobilized DNA probe that hybridizes with the target sequence. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye sulforhodamine B. The amount of liposomes captured in the detection zone can be read visually or quantified with a hand-held reflectometer. The biosensor can detect as little as 1 fmol target mRNA (1 nmol L(-1)). Specificity analysis revealed no cross-reactivity with 11 organisms tested, among them closely related species such as B. cereus, B. megaterium, B. subtilis, B. thuringiensis, Lactococcus lactis, Lactobacillus plantarum, and Chlostridium butyricum. Also, no false positive signals were obtained from nonviable spores. We suggest that this inexpensive biosensor is a viable option for rapid, on-site analysis providing highly specific data on the presence of viable B. anthracis spores.
The nucleotide sequence of an almost complete cDNA copy of chalcone synthase mRNA from cultured parsley cells (Petroselinum hortense) has been determined. The cDNA copy comprised the complete coding sequence for chalcone synthase, a short A-rich stretch of the 5' non-coding region and the complete 3' non-coding region including a poly(A) tail. The amino acid sequence deduced from the nucleotide sequence of the cDNA is consistent with a partial N-terminal sequence analysis, the total amino acid composition, the cyanogen bromide cleavage pattern, and the apparent mol. wt. of the subunit of the purified enzyme.
Viral and fungal infections and some cancers may be described as diseases that are characterized by the expression of certain unwanted proteins. They could be termed induced genetic disorders, with induction provided by mutation or infection. A comprehensive method to inactivate injurious genes based on their nucleic acid sequences has the potential to provide effective antiviral and anticancer agents with greatly reduced side effects. We describe a chemical approach to such gene-specific pharmaceutical agents. Our initial efforts have been to develop new chemical reagents that can carry out catalytic destruction of specific mRNA sequences. We chose hydrolysis as a chemical means of destruction, because hydrolysis is compatible with living cells. Our sequence-specific catalytic RNA hydrolysis reagents may be described as functional ribozyme mimics. Reactivity is provided by small-molecule catalysts, such as metal complexes. Specificity is provided by oligonucleotide probes. Here we report initial results on the sequence-specific, hydrolytic cleavage of mRNA from the HIV gag gene, using a ribozyme mimic. The reagent is composed of a terpyridylCu(II) complex for cleavage activity and an oligonucleotide for sequence specificity.
Four distinct kallikrein-related mRNAs (PS, S1, S2, and S3), encoded by members of a multigene family, are selectively expressed in various combinations in several rat tissues. Although closely related along most of the mRNA sequence, the four mRNAs can be selectively detected with synthetic oligonucleotide probes complementary to highly variable mRNA subregions. PS mRNA, which encodes an enzyme with true kallikrein activity, is present at high levels in the submaxillary gland, pancreas, and kidney. S1 mRNA, which encodes an enzyme similar to the PS kallikrein, is detected only in the submaxillary gland and is present at one-fifth the PS mRNA level. S2 mRNA, which encodes the enzyme tonin, is present in the submaxillary gland at half the PS mRNA level and at a slightly higher level in the prostate. S3 mRNA, which encodes an enzyme very similar to tonin, is present in the submaxillary gland at one-tenth the PS mRNA level and in the prostate at about the same level as tonin mRNA.
Hormones exert powerful effects on reproductive physiology by regulating gene expression. Recent discoveries in hormone action emphasize that regulation of gene expression is not restricted to their alterations of the rate of gene transcription. On the contrary, hormonal effects on the stability of a specific mRNA can profoundly alter its steady-state concentration. The mRNAs encoding hormone receptors are commonly regulated by their own hormones to create autoregulatory feedback loops. Negative and positive autoregulatory feedback loops serve to limit or augment hormonal responses, respectively. After introducing the topics of mRNA degradation and regulated stability, this review focuses on steroid hormone effects on mRNA stabilities. Autoregulation of the mRNAs encoding estrogen, progesterone, androgen, and glucocorticoid receptors by the steroid hormones in reproductive tissues is discussed. In addition, steroid hormone effects on the stabilities of many other mRNAs that are important to reproductive biology are reviewed. These include mRNAs that encode gonadotropin hormones, integrins, growth factors, and inflammatory response proteins. Through these posttranscriptional effects, steroid hormones impact the expression of a large population of genes. Studies of the molecular mechanisms of hormonally regulated mRNA stabilities continue to identify critical mRNA sequence elements and their interactions with proteins. Increased understanding of how hormones affect mRNA stability may yield novel approaches to the therapeutic control of hormone effects, including those essential to reproductive physiology in animals.