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PubMed2004

The multiassembly problem: reconstructing multiple transcript isoforms from EST fragment mixtures.

Recent evidence of abundant transcript variation (e.g., alternative splicing, alternative initiation, alternative polyadenylation) in complex genomes indicates that cataloging the complete set of transcripts from an organism is an important project. One challenge is the fact that most high-throughput experimental methods for characterizing transcripts (such as EST sequencing) give highly detailed information about short fragments of transcripts or protein products, instead of a complete characterization of a full-length form. We analyze this "multiassembly problem"-reconstructing the most likely set of full-length isoform sequences from a mixture of EST fragment data-and present a graph-based algorithm for solving it. In a variety of tests, we demonstrate that this algorithm deals appropriately with coupling of distinct alternative splicing events, increasing fragmentation of the input data and different types of transcript variation (such as alternative splicing, initiation, polyadenylation, and intron retention). To test the method's performance on pure fragment (EST) data, we removed all mRNA sequences, and found it produced no errors in 40 cases tested. Using this algorithm, we have constructed an Alternatively Spliced Proteins database (ASP) from analysis of human expressed and genomic sequences, consisting of 13,384 protein isoforms of 4422 genes, yielding an average of 3.0 protein isoforms per gene.

Algorithms↗

PubMed1989

The primary structure of human liver type phosphofructokinase and its comparison with other types of PFK.

The complete mRNA sequence of the human liver-type phosphofructokinase (hPFKL) was determined. The sequence included 55 nucleotides of 5' and 515 of 3' noncoding regions, as well as 2,337 nucleotides encoding the 779 amino acids of the hPFKL. Extensive similarity (approximately 90%) in the coding region was observed between the hPFKL and the mouse PFKL, whereas the degree of similarity between different types of PFK, i.e., hPFKL and human muscle-type PFK (hPFKM), was merely 68%. Nevertheless, striking similarity between these different types of PFK was noticed when the amino acid residues creating the various active sites of the enzyme were compared. Human PFK L- and M-specific probes were constructed and used to quantitate the mRNA levels in fetal and adult brains and fetal liver. It was found that while relative amount of PFKL mRNA in adult brain was one-fourth of that detected in fetal brain the level of PFKM mRNA in adult brain was slightly higher than in fetal tissue, suggesting that PFK expression might be controlled at the transcriptional level.

Amino Acid Sequence↗

PubMed1975

Estrogen induction of ovalbumin mRNA: evidence for transcription control.

Estrogen induces the synthesis and accumulation of the specific messenger RNA for the egg white protein ovalbumin. The messenger RNA has been purified to apparent homogeneity on a preparative scale and utilized to synthesize a radioactive complementary DNA copy. This complementary DNA probe was first used to reveal that although ovalbumin constitutes 60% of the total protein of chick oviduct the gene which codes for the ovalbumin nRNA is represented only once in each haploid genome: The induction of genetranscription and subsequent accumulation of ovalbumin mRNA during estrogen-mediated tissue differentiation was also investigated. Ovalbumin mRNA sequences were quantifiedusing the complementary DNA probe and by an in vitro heterologous translation system. Similar experiments were performed using chicks which were withdrawn from hormone treatment and then given a single injection of estrogen. The data suggest pure transcriptional control for the mechanism by which estrogen regulates the synthesis of the tissuespecific protein ovalbumin. Finally, several in vitro translation systems are comparedwith respect to their usefullness to assess the effects of hormones on mRNA production. It is concluded that the protein synthesis system derived from wheat germ offers thegreatest advantages for initial studies.

Animals↗

PubMedDate not supplied

Molecular cloning of German cockroach (Blattella germanica) allergens.

Allergens produced by cockroaches (CRs) are an important cause of IgE antibody responses and asthma. Using molecular cloning and nucleic acid hybridization techniques, we have identified and sequenced several important allergens produced by the German CR (Blattella germanica) and studied their expression in the American CR (Periplaneta americana). Principal allergens include Bla g 2 (36-kD protein) and Bla g 4 (21-kD protein), to which 60-70% of CR-allergic patients make IgE antibodies. Bla g 2 is only expressed by B. germanica, whereas DNA encoding Bla g 4 is present in P. americana, but is not transcribed into mRNA. Sequence homology searches have identified Bla g 2 as an aspartic protease and Bla g 4 as a calycin. Other CR allergens that have been cloned include a glutathione transferase and a troponin. These studies will enable recombinant allergens to be expressed and used to investigate the role of CR allergens in asthma.

Allergens↗

PubMed1976

Initial trp operon sequence in Escherichia coli is transcribed without coupling to translation.

The transcription of the "leader" region (Bronson et al., 1973) of the trp operon in Escherichia coli was studied in normal mutants which delete most of the operator-distal region of the operon [a deletion strain (trp OAEG) retaining only about one third of the "leader" region and two deletion strains (trpOAE14 and trpOAE2) retaining the whole "leader" region and an initial portion of the trpE], as well as in a strain with an intact trp operon, but with a temperature-sensitive lesion in ribosomal protein factor EFTs (strain HAK88). In these deletion mutants, mRNA molecules corresponding to the "leader" region were detected as most of the trp-specific mRNA. Less inhibition of transcription, of the promoter-proximal portion of the trp "leader" region than that of more distal genes of the operon, was found in chloramphenicol-treated cells of strain trpOAE14. It was also observed that transcription of the initial one third portion of the "leader" region was not repressed by tryptophan in strains trpOAE6 and trp OAE14. A similar effect of a translation block on transcription of the distal part of the "leader" region was observed with strain HAK88 at the nonpermissive temperature. In sedimentation analysis of polyribosomes containing the trp mRNA molecules from the deletion mutants, trp mRNA from strain trpOAE14 was found in monosomes and small polyribosomes, whereas the majority of the trp mRNA from strain trpOAE6 was found joined to a single ribosome or ribosomal subunit. These results suggest that ribosomes bind in vivo to a site(s) located in the middle of the "leader" mRNA sequence, and that the initial transcription of the trp operon does not require any connection to functional translational machinery, while continuation of RNA synthesis beyond a first ribosome binding site seems indispensably coupled to ribosome function.

DNA, Bacterial↗

PubMedDate not supplied

Ecto-ATPase mRNA is regulated by FSH in Sertoli cells.

A putative messenger RNA (mRNA) sequence, designated C8, that was up-regulated in Sertoli cells prepared from hypophysectomized rats treated with testosterone, was isolated from a Sertoli cell complementary DNA (cDNA) library. The coding region of C8 exhibited 99% identity with rat brain ecto-ATPase and expressed a 60-kilodalton protein following in vitro transcription/translation. Transfection of COS7 cells with C8 cDNA resulted in a marked increase in Ca2+- and Mg2+-dependent ATPase activity in both whole cells and cell homogenates, which is consistent with localization of this enzyme in the plasma membrane. C8 ecto-ATPase steady state mRNA levels were increased within 6 hours and for 3 day, by follicle-stimulating hormone (FSH) in Sertoli cells but not in peritubular cells. In contrast, dibutyryl-cyclic adenosine monophosphate (cAMP) increased ecto-ATPase in both Sertoli and peritubular cells. Testosterone had no significant effect under these conditions. These data indicate that ecto-ATPase mRNA is positively regulated by FSH in Sertoli cells and by cAMP in both Sertoli and peritubular cells. This enzyme may play a role in the control of extracellular signaling by ATP, adenosine, or both in the cells of the seminiferous epithelium.

Adenosine Triphosphatases↗

PubMed1995

Functional characteristics of the maize RNA-binding protein MA16.

The maize RNA-binding protein MA16 is a non-ribosomal nucleolar protein widely distributed in different maize tissues. We have previously shown that the MA16 protein binds preferentially to guanosine- and uridine-rich sequences. As a step towards the identification of specific targets with which MA16 interacts within the cell, we investigated the RNA-binding affinities and several other aspects of the protein by using binding assays and immunochemistry. The MA16 protein showed a wide spectrum of RNA-binding activities with lower affinities to several RNAs that was salt and heparin-sensitive indicative of electrostatic interactions, and higher affinities to particular RNAs including rRNA and translatable mRNA sequences. Among the RNAs found associated with MA16 protein was that encoding MA16 itself. This observation raises the possibility that MA16 gene expression could be self-regulated. Immunoprecipitation studies showed that in vivo MA16 was phosphorylated and that MA16 interacts with RNAs through complex association with several proteins. These results suggest that both phosphorylation and interaction with other proteins may be involved in determining RNA-binding specificities of MA16 in the cell.

Heat-Shock Proteins↗

PubMed2000

Identification of human estrogen-inducible transcripts that potentially mediate the apoptotic response in breast cancer.

Hormone manipulation has been used for several decades with the purpose of inducing breast cancer regression. On the one hand, hormone ablation and antiestrogen administration were used on the rationale that estrogens induce proliferation of their target cells. Before the advent of the antiestrogen tamoxifen, on the other hand, the estrogen agonist DES was used to obtain clinical remissions. The rationale for the use of diethylstilbestrol (DES) was totally empirical. In fact, the efficacy of both treatments was comparable. A mechanistic explanation for estrogen-induced regression is urgently needed in order to provide a rationale for its use in therapeutic fields, and to develop markers to identify this phenotype in order to recognize responsive tumors. In this report, we use E8CASS cells (a MCF7 variant) as a model to study estrogen-mediated regression. The proliferation rate of E8CASS cells is decreased by estrogens. In order to isolate mRNA sequences induced by estradiol, a subtracted library was prepared from E8CASS cells grown in the presence and absence of estrogens. Twenty nine differentially expressed unique sequences were found. Seven of them were homologous to known genes, 12 of them were homologous to expressed sequence tags (EST), and 10 sequences had no homologues in the databases. The two sequences showing the highest induction by estradiol (E9 and E43) were chosen for further analysis. The sequence of the E43 coding region has 96% homology to the bovine actin2 gene and 100% identity to bovine actin2 protein, and it is homologous to the human actin-related protein 3 (Arp3). It has been suggested that Arp3 is involved in actin nucleation. The phenotype of E8CASS cells is clearly affected by estrogen treatment. It is likely that E43 may be involved in these morphological changes. The E9 cDNA is a putative zinc-finger protein of the PHD family of transcriptional transactivators. A member of this family, Requiem, is involved in apoptosis. The E9 mRNA is highly expressed in E8CASS cells treated with estrogens, a treatment which results in decreased proliferation rate and increased DNA degradation. This correlation suggests that E9 may be a mediator of estrogen-induced regression of breast cancer.

Actins↗

PubMed2005

Expression of the plasma prekallikrein gene: utilization of multiple transcription start sites and alternative promoter regions.

The plasma prekallikrein gene is expressed in many different human tissues at distinctly different levels and therefore tissue-specific control of the gene transcription is likely. In this study we demonstrate that transcription of the plasma prekallikrein gene can be initiated at multiple sites, for which at least four different promoters are utilized. A comparison of the genomic and mRNA sequences of mouse plasma prekallikrein revealed that the sequence segment that was formerly regarded as the first exon of the mouse plasma prekallikrein gene consists of three exons, with the first exon localized 14.2 kbp upstream of the translation start. For the rat and human plasma prekallikrein genes, in silico analysis suggested an analogous exon-intron organization. Determination of the transcription start sites showed that in both mouse and human, the proximal and distal regions could be utilized for transcription initiation; however, the proximal region is preferred. A deletion mutation analysis of the proximal promoter region using a 1.7-kbp segment revealed a strong activating region immediately upstream of the known mRNA, followed by both a modest repressor and an enhancer region.

5' Untranslated Regions↗

PubMed1982

Codon usage in bacteria: correlation with gene expressivity.

The nucleic acid sequence bank now contains over 600 protein coding genes of which 107 are from prokaryotic organisms. Codon frequencies in each new prokaryotic gene are given. Analysis of genetic code usage in the 83 sequenced genes of the Escherichia coli genome (chromosome, transposons and plasmids) is presented, taking into account new data on gene expressivity and regulation as well as iso-tRNA specificity and cellular concentration. The codon composition of each gene is summarized using two indexes: one is based on the differential usage of iso-tRNA species during gene translation, the other on choice between Cytosine and Uracil for third base. A strong relationship between codon composition and mRNA expressivity is confirmed, even for genes transcribed in the same operon. The influence of codon use of peptide elongation rate and protein yield is discussed. Finally, the evolutionary aspect of codon selection in mRNA sequences is studied.

Amino Acid Sequence↗

PubMed1984

Evolution of the human sarcomeric-actin genes: evidence for units of selection within the 3' untranslated regions of the mRNAs.

The complete 3' untranslated region (3'UTR) sequence of the human skeletal-actin gene has been compared with the corresponding regions of the rat and chicken skeletal-actin genes. This comparison reveals that the skeletal-actin 3'UTR is composed of conserved and nonconserved segments. By using genomic Southern transfer blots and thermal stability (Tm) measurements, we found that the cardiac-actin gene 3'UTR also consists of conserved and nonconserved segments. Comparison of human and Xenopus laevis cardiac-actin mRNA sequences confirms the presence of a region of high similarity in the 3'UTR. We conclude that subsegments of the 3'UTRs of both skeletal- and cardiac-actin genes of birds and mammals are under considerable selective pressure. This suggests that these conserved sequences may have functional roles in actin-gene expression or regulation, and that these roles might be different for each actin isoform.

Actins↗

PubMed2004

Analysis of variation in mouse TPMT genotype, expression and activity.

Although the mouse has great potential for pharmacogenomic discovery, little is known about variation in drug response or genetic variation in pharmacologically relevant genes between inbred mouse strains. We therefore assessed variation in gene sequence, mRNA expression and protein activity of thiopurine methyltransferase (TPMT) in multiple inbred mouse strains. TPMT activity was measured by high-performance liquid chromatography detection of 6-MMP produced by incubation of liver homogenates with 6-MP. Genetic variation was assessed by resequencing and single nucleotide polymorphism (SNP) genotyping using pyrosequencing technology. mRNA expression was measured by real-time polymerase chain reaction. We observed an almost five-fold variation in TPMT activity, with strains falling into distinct low and high activity groups. This pattern of TPMT activity was highly correlated with expression of TPMT mRNA among strains, and high TPMT expression is dominant in F1 hybrids. To correlate genotype with phenotype, 29 SNPs and one insertion/deletion were genotyped throughout the TPMT gene and upstream 10 kb. Only two haplotypes were observed across all 30 polymorphisms, corresponding to the low and high activity groups. These results suggest that differential mouse TPMT activity is due to variation in mRNA expression. In addition, the identified pattern of low haplotype diversity suggests that the mouse is likely to be useful for pharmacogenomic discovery by associating haplotype blocks with drug response phenotypes among inbred strains.

Amino Acid Sequence↗

PubMedDate not supplied

Translational bypassing: a new reading alternative of the genetic code.

The translation of the genetic code, once thought to be rigid, has been found to be quite flexible, and several alternatives in its reading have been described. An unusual alternative is translational bypassing, a frameshift event where the transition from frame 0 to another frame occurs by translational bypassing of an extended region of the mRNA sequence rather than by slippage past a single nucleotide, as has been described for most examples of frameshifting. Translational bypassing has been characterized in two cases, T4 gene 60 coding for a topoisomerase subunit and in a trpR-lac'Z fusion. The latter was discovered in our laboratory, and the unique bypass mechanism is investigated further in this study. Using a trpR+1-lac'Z fusion system, we show that the Gln codon at the beginning of lacZ end at the 3' side of the gap is required for bypassing to occur. The Gln codon is part of an mRNA segment that can (potentially) base pair with a segment at the 5' and of Escherichia coli 16S rRNA. A model of trpR+1-lac'Z bypassing is suggested in which the untranslated region of the mRNA is looped out through base pairing between a segment in the 5' end of the 16S rRNA and two sites in the mRNA. Translational bypassing is a newly discovered mechanism of gene expression, and trpR is the first cellular gene identified in which such a mechanism could operate. The understanding of this mechanism and its associated signals may be considered a paradigm for the expression of other genes by this alternative reading of the genetic code.

Bacteriophage T4↗

PubMed1997

Suppression of anchorage-independent growth and matrigel invasion and delayed tumor formation by elevated expression of fibulin-1D in human fibrosarcoma-derived cell lines.

Using differential display, we identified an mRNA that is markedly down-regulated in cell line 6A/SB1, derived from a fibrosarcoma formed in an athymic mouse following injection of carcinogen-transformed MSU-1.1 cells. The nontumorigenic parental cell strain, MSU-1.1, expresses high levels of this mRNA. Sequencing of the corresponding cDNA fragment revealed that it corresponded to an expressed sequence tag, which ultimately led to its identification as the fibulin-1D gene. Fibulin-1 is a cysteine-rich, calcium-binding extracellular matrix and plasma protein, which has four isoforms, A-D, derived from alternative splicing. Northern and Western blotting analysis of 16 cell lines established from tumors formed in athymic mice by MSU-1.1-derived cell strains independently transformed in culture showed that 44% exhibited low level or lack of expression of fibulin-1D mRNA and protein. In a similar analysis of 15 malignant cell lines derived from patients, 80% showed low level or no expression. To study the role of fibulin-1D in transformation, we transfected 6A/SB1 cells and a human fibrosarcoma-derived cell line (SHAC) with a fibulin-1D cDNA expression construct. Transfectants displaying high levels of fibulin-1D were isolated and characterized. Elevated expression of fibulin-1D led to reduced ability to form colonies in soft agar and reduced invasive potential as tested in a matrigel in vitro invasion assay. Furthermore, expression of fibulin-1D resulted in a markedly extended latency in tumor formation in athymic mice. These results indicate that low expression of fibulin-1D plays a role in tumor formation and invasion.

Animals↗

PubMed1978

The precursor of mouse beta-globin messenger RNA contains two intervening RNA sequences.

We have investigated the locations of the poly(A), the mRNA-specific sequences and the RNA sequences that are eventually cleaved from the 1860 nucleotide precursor of mouse beta-globin mRNA. Biochemical and electron microscopic data demonstrate that there are two intervening RNA moieties in the precursor which separate the beta-globin mRNA sequences into three portions containing 480, 205 and 155 nucleotides. One of the two intervening RNA moieties contains 780 nucleotides. The size of the smaller intervening RNA has not been determined precisely, but it is 125 nucleotides or less. The largest mRNA-specific fragment is derived from the 3' terminus of the precursor, and contains the 3' terminal poly (A) and 330 mRNA-specific transcribed nucleotides. At least one, and probably both, intervening RNAs occur within the coding portion of the mRNA sequences. The larger of the intervening RNAs is located next to the 480 nucleotide mRNA-specific fragment, and the smaller intervening RNA is located between the 205 and 155 nucleotide mRNA-specific fragments. These experiments are consistent with the notion that the intervening sequences in the DNA of mouse beta-globin genes are transcribed into the mRNA precursor and are excised from the RNA by post-transcriptional events.

Animals↗

PubMed1997

Positional preferences of polypurine/polypyrimidine tracts in Saccharomyces cerevisiae genome: implications for cis regulation of gene expression.

The complete genome of the baker's yeast S. cerevisiae was analyzed for the presence of polypurine/polypyrimidine (poly[pu/py]) repeats and their occurrences were classified on the basis of their location within and outside open reading frames (ORFs). The analysis reveals that such sequence motifs are present abundantly both in coding as well as noncoding regions. Clear positional preferences are seen when these tracts occur in noncoding regions. These motifs appear to occur predominantly at a unit nucleosomal length both upstream and downstream of ORFs. Moreover, there is a biased distribution of polypurines in the coding strands when these motifs occur within open reading frames. The significance of the biased distribution is discussed with reference to the occurrence of these motifs in other known mRNA sequences and expressed sequence tags. A model for cis regulation of gene expression is proposed based on the ability of these motifs to form an intermolecular triple helix structure when present within the coding region and/or to modulate nucleosome positioning via enhanced histone affinity when present outside coding regions.

Chromosome Mapping↗

PubMed1978

Prolactin induction of casein mRNA in organ culture. A model system for studying peptide hormone regulation of gene expression.

The peptide hormone, prolactin, when added to organ explants of rat mammary gland, rapidly (within 1 h) induced the accumulation of casein mRNA. Casein mRNA sequences, as determined by hybridization with a specific cDNA probe, were shown to increase for up to 48 h after prolactin addition. The magnitude of this response was dependent upon the day of pregnancy at which the tissue was placed in culture. Maximal levels of induction (as great as 45-fold) were obtained using tissue from 15-day pregnant rats. Further data indicate that two steroid hormones, hydrocortisone and progesterone, were able to modulate the prolactin-induced accumulation of casein mRNA. The continuous presence of hydrocortisone was not necessary for prolactin induction of casein mRNA. However, the presence of hydrocortisone was required for maximal accumulation of casein mRNA. The induction of casein mRNA by prolactin was inhibited in a dose-dependent manner by the simultaneous addition of progesterone to the organ culture. Thus, hydrocortisone appears to potentiate the prolactin induction of casein mRNA, whereas progesterone is able to prevent casein mRNA accumulation. Since mammary gland organ culture is performed in a serum-free, chemically defined medium, this system allows a detailed examination of the mechanims by which a peptide hormone regulates the rapid accumulation of a specific mRNA.

Animals↗

PubMed1996

Structure of the C-terminal end of the nascent peptide influences translation termination.

The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA. For the penultimate amino acid (-2 location), charge and hydrophobicity are important. For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA). The influence of the -1 and -2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes.

Amino Acid Sequence↗