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PubMed1982

Rapid changes in the concentration of phosphoenolpyruvate carboxykinase mRNA in rat liver and kidney. Effects of insulin and cyclic AMP.

Starvation and diabetes both caused a marked increase in the concentration of hepatic phosphoenolpyruvate caroboxykinase mRNA while the administration of insulin to diabetic rats or refeeding glucose to starved animals caused a marked reduction in the levels of enzyme mRNA as measured by hybridization using a cDNA probe.l The Administration of dibutyryl cAMP to a starved-refed cat caused an 8-fold induction of phosphoenolpyruvate carboxykinase mRNA in 1 h. Triamcinolone plus acidosis induced the levels of enzyme mRNA in kidney 3-fold within 6 h, however, starvation for 24h had only marginal effects. In all of the above conditions, the levels of phosphoenolpyruvate carboxykinase mRNA measured by hybridization assay agreed well with the relative levels of translatable mRNA for the enzyme. The half-time of phosphoenolpyruvate carboxykinase mRNA, determined after the administration of either alpha-amanitin or cordycepin to starved animals, was approximately 40 min. However, cycloheximide either alone or together with cordycepin, not only prevented the decrease in phosphoenolpyruvate carboxykinase mRNA sequence abundance, but induced it 2-fold. Cycloheximide itself, when injected into 21-day fetal rats in utero caused an induction of enzyme mRNA equal to that noted when dibutyryl cAMP was administered. The mRNA for phosphoenolpyruvate carboxykinase is approximately 2.8 kb in length, but nuclei from the livers of diabetic rats contain a number of putative precursor RNA species for the enzyme, up to 6.5 kb in size, all containing a poly(A) tail. Two hours after refeedng glucose to a starved rat, these nuclear RNA species could no longer be detected by hybridization to our cDNA probe.

Animals↗
PubMed1984

Specific stimulation of actin gene transcription by epidermal growth factor and cycloheximide.

Stimulation of quiescent AKR-2B mouse embryo cells with epidermal growth factor (EGF) results in a rapid and specific induction of actin mRNA sequences. These mRNAs include those coding for both beta- and gamma-cytoskeletal, but not alpha-skeletal muscle, actin isotypes. Elongation of nascent RNA chains in isolated nuclei (run-off transcription) demonstrates that the mRNA accumulation is preceded by an increase in actin gene transcription. This increase is transient, however, and is followed by a rapid attenuation of transcriptional activity. An inhibitor of protein synthesis, cycloheximide, was also found to induce beta- and gamma-actin mRNA accumulation. Furthermore, the simultaneous addition of EGF and cycloheximide produced a synergistic effect on actin sequences in both steady-state nuclear and polysomal RNA. Run-off transcription experiments demonstrate that this synergistic effect results from an increase in the magnitude and duration of actin gene transcription. It is also specific in that alpha-tubulin gene transcription is not similarly affected. These data suggest the existence of a specific labile repressor of actin gene transcription.

Actins↗
PubMed1981

Hormonally regulated mammalian gene expression: steady-state level and nucleotide sequence of rabbit uteroglobin mRNA.

Uteroglobin is a protein that is synthesized in large quantities by the rabbit uterine endometrial cells and secreted into the uterine lumen around the time of implantation of the developing blastocysts. The protein is also synthesized constitutively at a low level in the lung. In the uterus, synthesis of the protein is induced by progesterone but repressed by estradiol; whereas in the lung, it is not hormonally responsive. Using a full-length cDNA clone, we have established the nucleotide sequence of uteroglobin mRNA and have determined its levels in uterus and lung during early pregnancy. The clone, pUG617, contains all but 24 nucleotides at the 5' untranslated region of the structural gene. To establish the full mRNA sequence, we isolated a 5' end-labeled DNA fragment from pUG617 and extended its length using reverse transcriptase after hybridization with uterine poly(A)-containing RNA. The 5'-terminal sequence of uteroglobin mRNA was established by sequencing the extended DNA fragment. The nucleotide sequence of the peptide-coding portion of the gene has resolved some previously reported discrepancies in the amino acid sequence of the mature protein and those in the signal peptide. By comparison of sequences with a partial uteroglobin cDNA clone isolated by another laboratory, a polymorphic nucleotide at position 246 of the gene has been identified, where a G-A transition has caused an amino acid substitution from aspartic acid to asparagine at residue 46 of the mature protein. Analysis of steady-state RNA levels in the uterus has shown that the induction and repression of uteroglobin synthesis during early pregnancy is the result of accumulation and depletion of its mRNA, respectively. During the same period in the lung, no consistent changes in uteroglobin mRNA level were evident, reflecting the constitutive levels of the protein in this tissue.

Amino Acid Sequence↗
PubMed2004

Effective inhibition of Rta expression and lytic replication of Kaposi's sarcoma-associated herpesvirus by human RNase P.

Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a nucleic acid-based gene interference approach to knock-down gene expression. Unlike other strategies, such as antisense oligonucleotides, ribozymes, and RNA interference, the RNase P-based technology is unique because a custom-designed EGS molecule can bind to any complementary mRNA sequence and recruit intracellular RNase P for specific degradation of the target mRNA. In this study, we demonstrate that the RNase P-based strategy is effective in blocking gene expression and growth of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), the causative agent of the leading AIDS-associated neoplasms, such as KS and primary-effusion lymphoma. We constructed 2'-O-methyl-modified EGS molecules that target the mRNA encoding KSHV immediate-early transcription activator Rta, and we administered them directly to human primary-effusion lymphoma cells infected with KSHV. A reduction of 90% in Rta expression and a reduction of approximately 150-fold in viral growth were observed in cells treated with a functional EGS. In contrast, a reduction of <10% in the Rta expression and viral growth was found in cells that were either not treated with an EGS or that were treated with a disabled EGS containing mutations that preclude recognition by RNase P. Our study provides direct evidence that EGSs are highly effective in inhibiting KSHV gene expression and growth. Exogenous administration of chemically modified EGSs in inducing RNase P-mediated cleavage represents an approach for inhibiting specific gene expression and for treating human diseases, including KSHV-associated tumors.

Antiviral Agents↗
PubMed1995

Alternative splicing results in two different transcripts for H-protein of the glycine cleavage system in the C4 species Flaveria trinervia.

Alternative splicing is a well-known post-transcriptional regulatory mechanism in eukaryotic organisms but there are only very few reports on alternative splicing in plants. The analysis of cDNAs encoding H-protein of the glycine decarboxylase multi-enzyme complex from the C4 species Flaveria trinervia revealed the presence of two transcript populations that differ in the length of their coding regions by six nucleotides. Otherwise, including their 3' nontranslated region, they are identical. From a genomic Southern analysis and from the sequencing of several independent cDNA clones it is evident that both types of transcript are derived from a single-copy gene. This gene, FTgdcsH, has been cloned and sequenced. It comprises four short exons. The two alternative splice sites are located at the end of intron 1. The shorter transcript closely corresponds to published H-protein mRNA sequences from other organisms. The longer transcript encodes two additional alanine residues very close to the N-terminus of the mature H-protein. A quantification of the relative amounts of both transcripts in different organs revealed that, with 80-90% of the total H-protein mRNA, the alternative mRNA dominates in leaves whereas roots contain more of the 'correctly' spliced transcript. It is concluded that, in F. trinervia and with a distinct organ preference, alternative splicing leads to the synthesis of two different H-proteins of the glycine cleavage system.

Alternative Splicing↗
PubMed1997

Functional importance of RNA interactions in selection of translation initiation codons.

RNA base pairing between the initiation codon and anticodon loop of initiator tRNA is essential but not sufficient for the selection of the 'correct' mRNA translational start site by ribosomes. In prokaryotes, additional RNA interactions between small ribosomal subunit RNA and mRNA sequences just upstream of the start codon can efficiently direct the ribosome to the initiation site. Although there is presently no proof for a similar important ribosomal RNA interaction in eukaryotes, the 5' non-coding regions of their mRNAs and 'consensus sequences' surrounding initiation codons have been shown to be strong determinants for initiation-site selection, but the exact mechanisms are not yet understood. Intramolecular base pairing in mRNA and participation of translation initiation factors can strongly influence the formation of mRNA-small ribosomal subunit-initiator tRNA complexes and modulate translational activities in both prokaryotes and eukaryotes. Only recently has it been appreciated that alternative mechanisms may also contribute to the selection of initiation codons in all organisms. Although direct proof is currently lacking, there is accumulating evidence that additional cis-acting mRNA elements and trans-acting proteins may form specific 'bridging' interactions with ribosomes during translation initiation.

Base Sequence↗
PubMed1980

Restriction mapping of cDNA recombinants including the adult chicken and duck globin messenger sequences: a comparative study.

A comparison of the organization of six avian adult globin messenger sequences is based on previously reported recombinant duck adult globin cDNA plasmids (Therwath et al., 1980) and the actual construction and characterization of pBR322 recombinant plasmids including the beta and the normal alpha A and alpha D chicken adult globin mRNA sequences. Identification of the cloned DNA was performed using hybridization-selection under conditions permitting complete purification in one step of the three globin mRNAs, and translation of the corresponding mRNA. Orientation of the globin insert in the vector was determined, taking into account the computer prediction of the restriction sites based on the known amino acid sequences of the three globin chains (Roizès and Pelaquier, 1980) and those actually observed, and by identification of restriction fragments using 3'-specific probes. Identification, orientation and restriction mapping of these cloned DNAs reveals extensive homologies in organisation of beta sequences between duck and chicken, as well as among the alpha sequences in every two possible combinations.

Animals↗
PubMed1989

The VH gene sequences of anti-DNA antibodies in two different strains of lupus-prone mice are highly related.

The heavy and light chain V region sequences of an IgG anti-DNA autoantibody (PME77), derived from a lupus-prone (NZB x NZW)F1 mouse have been determined by mRNA sequencing. The V kappa gene segment belongs to the V kappa 1A gene sub-group and is found in several (NZB x NZW)F1 and MRL lpr/lpr anti-DNA antibodies, as well as in other antibodies of unrelated specificities. The VH gene segment appears to represent a unique gene or a subfamily of the large J558 VH gene family of the mouse, and is highly related to a germ-line sequence of a major anti-DNA idiotype (H130, IgM) of MRL mice. This anti-DNA-related VH segment has not been found, so far, to be expressed in antibodies with specificities for external or synthetic antigens; therefore, expression of such specificities may be regulated by powerful mechanisms of self tolerance in the healthy animal. In addition, both the heavy and light chain of the PME77 IgG antibody were found to contain somatic point mutations with a high ratio of replacement to silent mutations in complementarity determining regions. This IgM to IgG sequence relationship suggests an affinity maturation process, which is driven by the autoantigen.

Amino Acid Sequence↗
PubMed2000

Molecular beacons for DNA biosensors with micrometer to submicrometer dimensions.

Ultrasensitive molecular beacon (MB) DNA biosensors, with micrometer to submicrometer sizes, have been developed for DNA/RNA analysis. The fluorescence-based biosensors have been applied in DNA/ RNA detection without the need for a dye-labeled target molecule or an intercalation reagent in the testing solution. Molecular beacons are hairpin-shaped oligonucleotides that report the presence of specific nucleic acids. We have designed a surface-immobilizable biotinylated ssDNA molecular beacon for DNA hybridization at a liquid-solid interface. The MBs have been immobilized onto ultrasmall optical fiber probes through avidin-biotin binding. The MB DNA biosensor has been used directly to detect, in real time, its target DNA molecules without the need for a competitive assay. The biosensor is stable and reproducible. The MB DNA biosensor has selectivity with single base-pair mismatch identification capability. The concentration detection limits and mass detection limits are 0.3 nM and 15 amol for a 105-microm biosensor, and 10 nM and 0.27 amol for a submicrometer biosensor, respectively. We have also prepared molecular beacon DNA biosensor arrays for simultaneous analysis of multiple DNA sequences in the same solution. The newly developed DNA biosensors have been used for the precise quantification of a specific rat gamma-actin mRNA sequence amplified by the polymerase chain reaction.

Actins↗
PubMed2000

Cloning of TC-1 (C8orf4), a novel gene found to be overexpressed in thyroid cancer.

A novel gene highly expressed in thyroid cancer, designated TC-1 (thyroid cancer-1), was cloned from suppression subtractive hybridization between papillary thyroid carcinoma and its surrounding normal thyroid tissue. Overexpression of TC-1 in thyroid cancer was confirmed in 15/16 paired samples by RT-PCR and Northern analysis. Ubiquitously expressed in human tissues, the TC-1 sequence showed no homology to any known gene, but matched a cluster of ESTs. After alignment of our sequence with the ESTs, the missing transcription start site was obtained by 5'-RACE and verified by primer extension analysis. The full-length mRNA sequence of 1327 bp has an open reading frame of 321 bp, which encodes a highly conserved protein. Three regulatory motifs were identified at the expected positions within 1 kb of the 5' flanking sequence obtained by genome walking. Using fluorescence in situ hybridization, TC-1 was localized to chromosome 8p11.2. The overexpression of TC-1 in papillary carcinoma suggests that it may have an important role in thyroid carcinogenesis.

Amino Acid Sequence↗
PubMed2006

Prediction of non-coding and antisense RNA genes in Escherichia coli with Gapped Markov Model.

A new mathematical index was developed to identify and characterize non-coding RNA (ncRNA) genes encoded within the Escherichia coli (E. coli) genome. It was designated the GMMI (Gapped Markov Model Index) and used to evaluate sequence patterns located at the separate positions of consensus sequences, codon biases and/or possible RNA structures on the basis of the Markov model. The GMMI was able to separate a set of known mRNA sequences from a mixture of ncRNAs including tRNAs and rRNAs. Consequently, the GMMI was employed to predict novel ncRNA candidates. At the beginning, possible transcription units were extracted from the E. coli genome using consensus sequences for the sigma70 promoter and the rho-independent terminator. Then, these units were evaluated by using the GMMI. This identified 133 candidate ncRNAs, which contain 29 previously annotated small RNA genes and 46 possible antisense ncRNAs. Furthermore 12 transcripts (including five antisense RNAs) were confirmed according to the expression analysis. These data suggests that the expression of small antisense RNAs might be more common than previously thought in the E. coli genome.

Computational Biology↗
PubMedDate not supplied

The elongating ribosome: structural and functional aspects.

We determined the positions and arrangements of RNA ligands within the ribosome with a new neutron-scattering technique, the proton-spin contrast-variation. Two tRNAs were bound to the ribosome in the pre-translocational and the post-translocational state. The mass centre of gravity of both tRNAs resides at the subunit interface of the body of the 30S subunit. Both tRNAs are separated by an angle of 50-55 degrees, and their mutual arrangement does not change during translocation. The mass centre of gravity moves by 13 +/- 3 A (1A = 0.1 nm) during translocation, corresponding well with the length of one codon. Using an RNase-digestion technique, the length of the mRNA sequence covered by the ribosome was determined to be 39 +/- 3 nucleotides before and after translocation. The ribosome moves like a rigid frame along the mRNA during translocation. In contrast, both tRNAs seem to be located on a movable ribosomal domain, which carries the tRNAs before, during, and after translocation, leaving the microtopography of the tRNAs with the ribosome unaltered. This conclusion was derived from an analysis of the contract patterns of thioated tRNAs on the ribosome. The results have led to a new model of the elongation cycle, which reinterprets the features of the previous "allosteric three-sites model" in a surprisingly simple fashion. Finally, a mutational analysis has identified a single nucleotide of the 23S rRNA essential for the peptidyltransferase activity.

Amino Acid Sequence↗
PubMed2002

NATH, a novel gene overexpressed in papillary thyroid carcinomas.

In this study a replica cDNA screening (RCS) approach to identify genes differentially expressed in papillary thyroid carcinomas (PTC) was used, as compared to non-neoplastic thyroid tissues. RCS is based on hybridization of radioactively labeled cDNA probes made from the biopsies to replica membranes with 15 000 clones from a PTC cDNA library. Among the genes overexpressed in PTC, and especially in clinically aggressive tumors with histologic evidence of poorly differentiated or undifferentiated areas, a novel gene named NATH was found. NATH has two mRNA species, 4.6 and 5.8 kb, both harboring the same open reading frame encoding a putative protein of 866 amino acids. The NATH protein is homologous to yeast N-acetyltransferase (NAT)1 and to mouse NARG1 (mNAT1) and contains four tetratricopeptide repeat (TPR) domains, suggesting that NATH may be part of a multiprotein complex. Overlapping RT-PCR fragments from several PTC biopsies confirmed the NATH mRNA sequence. Northern blots, semiquantitative RT-PCR experiments, TaqMan real-time RT-PCR experiments, and in situ hybridization verified the overexpression of NATH mRNA localized to tumor cells in PTC biopsies. NATH was expressed at a low level in most human adult tissues, including the normal thyroid gland. Increased NATH expression was seen especially in a Burkitt lymphoma cell line and in adult human testis. Recombinant in vitro expression showed that NATH protein was located mainly in the cytoplasm, and was present as a single protein band of the expected 105 kDa molecular weight. Heterologous expression of NATH in the papillary carcinoma cell line (NPA) and 293 cells did not alter the cellular proliferation rate. The biological function of NATH remains to be elucidated, but the overexpression in classic PTC and especially in poorly differentiated or undifferentiated components may indicate a function in the progression of papillary thyroid carcinomas.

Acetyltransferases↗
PubMed1979

High-molecular-weight nuclear polyadenylate-containing ribonucleic acid isolated from the lactating guniea-pig mammary gland contains milk-protein messenger ribonucleic acid sequences.

1. Steady-state poly(A)-containing nuclear RNA was isolated from the lactating guinea-pig mammary gland and analysed by sucrose-gradient centrifugation under denaturing conditions. 2. Nucleic acid-hybridization studies demonstrated the presence of small amounts of high-molecular-weight RNA species containing milk-protein mRNA sequences sedimenting at 25S. These were not found in the post-nuclear supernatant, where milk-protein sequences sedimented only between 12S and 15S; these were also the predominant species in the nuclear fraction. 3. The results are consistent with the transcription of milk-protein genes as high-molecular-weight precursor RNA species, 3-4 times as large as the active mRNA species isolated from the post-nuclear fraction.

Animals↗
PubMed1991

Differential translatability in vitro of multiple messenger RNAs encoding the beta-subunit of mouse thyrotropin.

The mouse TSH beta gene contains two start sites of transcription and exhibits alternative splicing among its first three exons, which encode 5'-untranslated mRNA sequences. Expression of the mouse TSH beta gene, therefore, gives rise to multiple mRNAs, each with a unique 5'-untranslated region. We have determined the relative translational efficiencies of these mRNAs in vitro, and we demonstrate that one of them directs the synthesis of a novel TSH beta presubunit. The four TSH beta mRNAs that are expressed from the down-stream transcription start site (TSS2) and the major mRNA derived from the up-stream start site (TSS1) were transcribed in vitro and translated in reticulocyte lysates and wheat germ extracts. The mRNA from TSS1 gave a novel TSH beta presubunit due to initiation of translation at an up-stream AUG unique to this mRNA. The novel presubunit contained a 17-amino acid NH2-terminal extension sequence, compared to the normal TSH beta presubunit, which is encoded by each of the mRNAs from TSS2. Despite the fact that the NH2-terminal extension sequence appeared to lack the characteristics of a signal peptide, the novel TSH beta presubunit was processed about 50% as efficiently by microsomal membranes as the normal presubunit, with glycosylation and cleavage by signal peptidase. There was an approximately 2-fold range in relative translatability among the different TSH beta mRNAs, and the mRNA encoding the novel TSH beta presubunit had the highest translational efficiency. Our data, therefore, suggest that the longer presubunit may be synthesized in vivo in significant amounts and give rise to a novel mature TSH beta subunit.

Amino Acid Sequence↗
PubMed1994

Sequence of a rabbit sperm zona pellucida binding protein and localization during the acrosome reaction.

The interaction of the mammalian spermatozoon with the oocyte's extracellular matrix or zona pellucida is a critical first step leading to successful fertilization. In this cell-extracellular matrix interaction it is the carbohydrate of the zona pellucida which serves as the sperm receptor and the surface of the spermatozoon which provides the lectin-like adhesion molecules. To better understand sperm-zona pellucida binding we have analyzed one specific zona binding protein (ZBP). This study has determined the mRNA sequence encoding a mammalian testis and sperm specific protein of 16,891 Da, which we have designated Sp17. Analysis of Sp17 revealed that the mRNA is present in rabbit, mouse, and human testes but not in any somatic tissue tested. In the rabbit, Sp17 is the 17-kDa member of the rabbit sperm autoantigen family of sperm specific autoantigens and is encoded by two mRNAs of 0.9 and 1.1 kb. Each mRNA has a unique 5' untranslated region but both have identical coding regions. The deduced amino acid sequence of the Sp17 ZBP showed several interesting features, including a similarity to the N-terminal of human testis cAMP-dependent protein kinase. Localization of Sp17 on live spermatozoa using antibodies to recombinant Sp17 or to the Sp17 peptide, G22C, revealed that the peptide backbone of Sp17 is inaccessible until the acrosome reaction begins. However, on paraformaldehyde fixed, acrosome intact spermatozoa, the peptide backbone is accessible to the antibodies which localize Sp17 to the apical surface. In the rabbit as well as other similar species in which the corona radiata (granulosa) cells adhere tightly to the zona pellucida and synthesize zona glycoproteins, the fertilizing spermatozoon may have already begun the acrosome reaction within the cumulus oophorus. Thus, the rabbit sperm surface would be modified to expose the Sp17 polypeptide during the final phase of cumulus passage and consequently Sp17 would be available for initial zona binding. The present study has also demonstrated that recombinant Sp17 can bind zona pellucida, dextran, and dextran sulfate.

Acrosome↗
PubMed1995

The sequence and genomic organization of the human type 2 angiotensin II receptor.

A human genomic DNA library was screened utilizing a human angiotensin II type 2 receptor (hAT2R) cDNA as a probe. Several positive clones were isolated and characterized. A comparison of the hAT2R cDNA sequence with the hAT2R genomic clone sequence suggests that the hAT2R gene is composed of three exons and spans at least 5 kb. Exons 1 and 2 encode for 5' untranslated mRNA sequence and exon 3 harbors the entire uninterrupted open reading frame of the hAT2R. Sequence analysis of the 5'-flanking region of the hAT2R gene demonstrates that it contains the typical sequence motifs found in many eukaryotic promoters. Interestingly, however, this promoter region also includes an interferon consensus sequence binding protein site (ICSBP) and a putative embryonal, long terminal repeat binding protein (ELP) site. The presence of these novel putative transcription factor binding sites suggests that this gene may be regulated in a unique manner.

Animals↗
PubMed1996

Characterization of the mouse Tdgf1 gene and Tdgf pseudogenes.

Cripto protein is a member of the "EGF family" of growth factors present in colon tumors and in human and mouse undifferentiated teratocarcinoma cells. During gastrulation in the mouse, cripto-encoding transcripts are expressed in the forming mesoderm and later in the truncus arteriosus of the developing heart. As a necessary step prior to investigating the in vivo role of cripto through gene disruption, we have isolated all the genomic cripto-related sequences in the mouse. One gene (Tdgf1) and two pseudogenes (Tdgf2 and Tdgf3) have been isolated and characterized. The mouse Tdgf1 (coding for cripto), like the human gene, is divided into six exons. Comparison of the human and mouse genomic sequences reveals that mouse exons 1 and 3 are shorter than the corresponding human exons. The pseudogene Tdgf2 corresponds to about 1 kb of the mRNA and contains five base substitutions in the coding region that represent both silent and replacement substitutions. The pseudogene Tdgf3 corresponds only to the coding portion of Tdgf. Many mutations have been introduced in this pseudogene, suggesting its early origin. Alignments of the Tdgf3, human and mouse mRNA sequences, shows that this pseudogene has retained the 33 nucleotides of the human exon 3 that are missed in the Tdgf1 gene. Taken together, these data suggest that Tdgf3 is derived from an ancestral gene and that the human and mouse genes are probably evolving separately.

3T3 Cells↗