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PubMed2005

Random sequencing of cDNA library derived from partially-fed adult female Haemaphysalis longicornis salivary gland.

A cDNA library was constructed from salivary glands of partially-fed adult female Haemaphysalis longicornis (hard tick). Randomly selected clones were sequenced and a total of 633 sequences were analyzed by bioinformatic programs. The sequences were grouped into 213 clusters, with each cluster being considered to be composed of mRNAs derived from the same gene or closely related genes. About 36% of the mRNA sequences showed significant similarity to known proteins in the non-redundant protein database by the NCBI blastx program and appeared to be coding for functional predicted proteins, whereas the remaining 64% had no similar sequences. Two thirds of the predicted proteins were annotated as basic cellular proteins (housekeeping proteins). Among the functional predicted protein sequences, other than the housekeeping proteins, several protease inhibitors including anticoagulants, two metalloproteases and a potential immunosuppressive protein could be identified. These proteins may play important roles during tick feeding and could be novel anti-tick vaccine candidates.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of Ig heavy chains.

Our prior analyses defined the cDNA sequence on part of the CH2 domain, the complete CH3 and CH4 domains, and the 3'-untranslated region of a catfish H chain. To complete the catfish H chain mRNA sequence, a primer-extended H chain cDNA library was constructed. Analysis of this library has resulted in the definition of full-length clones encoding a 61-bp 5' untranslated region, a 51-bp leader sequence, the V region and the complete CH1 and CH2 domains. The high similarity defined with other vertebrate V regions readily allowed the catfish sequence to be divided into FR and CDR regions. Sequence comparisons with mammalian VH and JH genes strongly suggest that the catfish V region is the product of multiple genes. Using a catfish VH cDNA probe, at least 25 different genomic VH members were defined. Because this probe does not hybridize with other full-length H chain cDNA clones, additional VH families will likely be defined in catfish. Phylogenetic sequence comparisons of the catfish C region domains indicated that the CH1 and CH4 were the most highly conserved. In addition several important features were defined in genomic Southern blot analyses of catfish DNA. Gene titration experiments established that the catfish CH gene is represented by a single genomic copy. This finding provides clear evidence that the genomic organization of H chain genes in catfish must be different from that defined in sharks and suggests that the phylogeny of single copy CH genes may have been established at the level of the bony fishes. It is also likely that there is an additional CH gene in catfish. This gene is also represented by a single genomic copy, and based upon its relative signal intensity when compared with the known CH gene it appears to share higher similarity with the known CH1 domain than it does with the CH2 domain.

Amino Acid Sequence↗
PubMed1975

Messenger RNA metabolism of animal cells. Possible involvement of untranslated sequences and mRNA-associated proteins.

The past several years have seen a virtual revolution in the study of eukaryotic mRNA. Among the notable recent achievements are the positive identification of mRNA precursors in HnRNA, the enumeration of the DNA sequences from which mRNA is transcribed, and the finding that mRNA in cultured cells is much more stable than was previously believed. One of most far-reaching discoveries has been the finding that mRNA in eukaryotes contains poly A. This discovery, aside from providing a powerful tool for mRNA isolation, has generated a large body of research into the properties and metabolism of poly A itself. In addition, the finding of a poly A-associated protein has given a renewed stimulus to the study of proteins associated with mRNA. This review is devoted to a discussion of these and related achievements, and some of their implications

Adenine↗
PubMed1993

Isolation and characterization of a cDNA encoding rat liver cytosolic epoxide hydrolase and its functional expression in Escherichia coli.

A cDNA of 1992 base pairs encoding the complete rat liver cytosolic epoxide hydrolase has been isolated using a polymerase chain reaction-derived DNA fragment (Arand, M., Knehr, M., Thomas, H., Zeller, H. D., and Oesch, F. (1991) FEBS Lett. 294, 19-22) known to represent the 3'-end of the cytosolic epoxide hydrolase mRNA. Sequence analysis revealed an open reading frame of 1662 nucleotides corresponding to 554 amino acids (M(r) = 62,268). The DNA sequence obtained did not display significant homology to the sequences of microsomal epoxide hydrolase or leukotriene A4 hydrolase or to any other DNA included in the EMBL Data Bank (release 32). On Northern blotting of rat liver RNA, a single mRNA species was detected that was strongly induced on treatment of the animal with fenofibrate, a potent peroxisome proliferator. The most significant structure of the deduced protein is a modified peroxisomal targeting signal (Ser-Lys-Ile) at the carboxyl terminus that is regarded to be responsible for the unusual dual localization of the cytosolic epoxide hydrolase in peroxisomes as well as in the cytosol. In addition, a leucine zipper-like motif was identified at the amino terminus. Its possible implication for the observed dimeric structure of cytosolic epoxide hydrolase is discussed. The isolated cDNA was expressed in bacteria to yield a catalytically active enzyme. Specific activity of the crude lysate obtained exceeded that of rat liver cytosols from maximally induced animals by a factor of 8.

Amino Acid Sequence↗
PubMed1987

Monoclonal BALB/c anti-progesterone antibodies use family IX variable region heavy chain genes.

Variable region nucleotide sequences and respective translated amino acid sequences for three heavy chains (DB3, 11/32 and 10/8) and two light chains (DB3 and 11/32) of monoclonal mouse IgG1 anti-progesterone antibodies have been determined by primer extension mRNA sequencing. The three VH regions exhibit the same rarely observed VH IX gene family and have greater than 88% homology between them. Two associated light chain sequences are 95% homologous and belong to the V kappa I group. The N-terminal twenty two amino acids of the kappa light chain of the third antibody 10/8 have been determined by automated protein sequencing and are identical to those of 11/32. Thus, these three monoclonal anti-progesterones derived from separate fusions all use VHIX-V kappa 1 gene combinations.

Amino Acid Sequence↗
PubMed1989

'Allelic' forms of immunoglobulin V genes in different strains of mice.

A VH gene (Ox1) has a major role in the early antibody response of several mouse strains to hapten phenyloxazolone (phOx). Antibodies that are coded by this gene are positive for idiotype 495. Idiotype-positive monoclonal antibodies originating from the early primary response of nine strains were partially sequenced (mRNA). All 21 antibodies were coded by this gene, most of them also by one VL gene, VKOx1(H3). Very few somatic mutations were found, and the germ-line sequence of the two genes in several strains can be predicted. Four 'alleles' of the VHOx1 gene have 99-99.7% sequence homology to each other. One allele was found in Igh allotype j strains CBA and C3H, another in allotype c strains DBA/2 and RF, the third in allotype f strain CE and the fourth in BALB/c, 129, A/J and RIII mice (allotypes a, e or g). The VKOx1(H3) gene has the same sequence in eight strains. RF mice do not use this gene for the anti-phOx response. Our data suggest that antibody responses are inherited to a considerable extent and that immunoglobulin V genes are as stable as other genes in evolution.

Alleles↗
PubMed1987

The AT spacers and the var1 genes from the mitochondrial genomes of Saccharomyces cerevisiae and Torulopsis glabrata: evolutionary origin and mechanism of formation.

Intergenic sequences represent 63% of the mitochondrial 'long' (85 kb) genome of Saccharomyces cerevisiae. They comprise 170-200 AT spacers that correspond to 47% of the genome and are separated from each other by GC clusters, ORFs, ori sequences, as well as by protein-coding genes. Intergenic AT spacers have an average size of 190 bp, and a GC level of 5%; they are formed by short (20-30 nt on the average) A/T stretches separated by C/G mono- to trinucleotides. An analysis of the primary structures of all intergenic AT spacers already sequenced (32 kb; 80% of the total) has shown that they are characterized by an extremely high level of short sequence repetitiveness and by a characteristic sequence pattern; the frequencies of A/T isostichs conspicuously deviate from statistical expectations, and exponentially decrease when their (AT + TA)/(AA + TT) ratio, R, decreases. A situation basically identical was found in the AT spacers of the mitochondrial genome (19 kb) of Torulopsis glabrata. The sequence features of the AT spacers indicate that they were built in evolution by an expansion process mainly involving rounds of duplication, inversion and translocation events which affected an initial oligodeoxynucleotide (endowed with a particular R ratio) and the sequences derived from it. In turn, the initial oligodeoxynucleotide appears to have arisen from an ancestral promoter-replicator sequence which was at the origin of the nonanucleotide promoters present in the mitochondrial genomes of several yeasts. Common sequence patterns indicate that the AT spacers so formed gave rise to the var1 gene (by linking and phasing of short ORFs), to the DNA stretches corresponding to the untranslated mRNA sequences and to the central stretches of ori sequences from S. cerevisiae.

Base Sequence↗
PubMedDate not supplied

Recent gene conversion involving bovine vasopressin and oxytocin precursor genes suggested by nucleotide sequence.

The nonapeptide hormones arginine vasopressin (AVP) and oxytocin (OT) are synthesized in the hypothalamus together with their carrier proteins, the neurophysins, as common polypeptide precursors. The organization of these precursors has been established by sequence determination of cloned bovine cDNAs encoding prepro-arginine vasopressin-neurophysin II (prepro-AVP-NPII) and prepro-oxytocin-neurophysin I (prepro-OT-NPI). When the mRNA sequences coding for the conserved middle part of the neurophysins were compared, we found that these sequences are not merely similar but identical. The primary structure of the chromosomal genes now determined shows that both genes, which appear to have arisen by a gene duplication, are split into three exons, each encoding a functional domain of the precursor polypeptide. Sequence comparison reveals that the stretch of sequence identity within the two mRNAs is probably the result of a gene conversion encompassing exon B, which encodes the conserved part of the neurophysins, and part of the preceding intron.

Amino Acid Sequence↗
PubMed2002

Cystic fibrosis transport regulator and its mRNA are expressed in human epidermis.

Cystic fibrosis transport regulator is a cAMP-dependent chloride channel protein. Normal (non cystic fibrosis) human epidermis stained positive for cystic fibrosis transport regulator as densely as did the eccrine sweat gland when three monoclonal antibodies for R (regulatory) and C (C-terminus) domains of cystic fibrosis transport regulator were used. All the layers of the epidermis took up staining uniformly. A peptide for C-epitope completely blocked the staining with monoclonal antibodies for C. Nested reverse transcription polymerase chain reaction of freshly isolated human epidermal fragments and the eccrine sweat glands amplified the cystic fibrosis transport regulator mRNA sequence derived from exons 13 and 14 to comparable extents. The 526 base pair antisense, but not sense, RNA probe derived from exons 10-13 stained cystic fibrosis transport regulator mRNA in both the epidermis and the sweat gland to a similar extent. In the epidermis, the cytoplasm of basal cells, stratum spinosum cells, and granular layer cells were all stained uniformly, but not corneocytes in the stratum corneum. In the sweat secretory coils, both clear and dark cells were stained but not the myoepithelium, with the dark cells staining more densely than the clear cells as in a previous study. In the duct, both luminal and basal ductal cells took up cystic fibrosis transport regulator staining uniformly but luminal cytoplasm of luminal ductal cells was devoid of cystic fibrosis transport regulator mRNA. Although the function of cystic fibrosis transport regulator in the epidermis is totally unknown, its recently proposed role as a universal regulator of a variety of cellular and membrane functions necessitates further studies on its regulation and function in health and disease.

Adult↗
PubMed1983

alpha-skeletal and alpha-cardiac actin genes are coexpressed in adult human skeletal muscle and heart.

We determined the actin isotypes encoded by 30 actin cDNA clones previously isolated from an adult human muscle cDNA library. Using 3' untranslated region probes derived from alpha-skeletal, beta- and gamma-actin cDNAs and from an alpha-cardiac actin genomic clone, we showed that 28 of the cDNAs correspond to alpha-skeletal actin transcripts. Unexpectedly, however, the remaining two cDNA clones proved to derive from alpha-cardiac actin mRNA. Sequence analysis confirmed that the two skeletal muscle alpha-cardiac actin cDNAs are derived from transcripts of the cloned alpha-cardiac actin gene. Direct measurements of actin isotype mRNA expression in human skeletal muscle showed that alpha-cardiac actin mRNA is expressed at 5% the level of alpha-skeletal actin. Furthermore, the alpha-cardiac actin gene expressed in skeletal muscle is the same gene which produces alpha-cardiac actin mRNA in the human heart. Of equal surprise, we found that alpha-skeletal actin mRNA accounts for about half of the total actin mRNA in adult heart. Comparison of total actin mRNA levels in adult skeletal muscle and adult heart revealed that the steady-state levels in skeletal muscle are about twofold greater, per microgram of total cellular RNA, than those in heart. Thus, in skeletal muscle and in heart, both of the sarcomeric actin mRNA isotypes are quite abundant transcripts. We conclude that alpha-skeletal and alpha-cardiac actin genes are coexpressed as an actin pair in human adult striated muscles. Since the smooth-muscle actins (aortic and stomach) and the cytoplasmic actins (beta and gamma) are known to be coexpressed in smooth muscle and nonmuscle cells, respectively, we postulate that coexpression of actin pairs may be a common feature of mammalian actin gene expression in all tissues.

Actins↗
PubMed1994

Complete sequence of rat MAP2d, a novel MAP2 isoform.

MAP2 proteins are among the major microtubule-associated proteins in the vertebrate nervous system. The high molecular weight proteins MAP2a, MAP2b and the low molecular weight protein MAP2c are encoded by distinct mRNAs generated by alternative splicing from the same primary transcript. MAP2c is primarily found in embryonic and neonatal brain. Using primers selected in the 5' and 3' non-coding regions of the rat MAP2c sequence and reverse transcription-coupled PCR from adult rat brain RNA, we have amplified and sequenced the entire coding region of a novel MAP2 isoform containing a 93 base pair insertion. Sequence analysis reveals that the additional exon encodes a domain that is highly homologous to repeats found in the 3' end of the coding region of MAP2, MAP4 and Tau mRNA sequences. In these microtubule-associated proteins, the domains encoded by the repeats are involved in microtubule binding and bundling.

Animals↗
PubMed1976

[Composition and variability of bases in degenerate and non-degenerate positions in codons of various types of mRNA].

Codon base frequencies have been studied in all published mRNA sequences of more than 50 codons. Bases are not "random" in any codon position. Variability in degenerate position III is of the same order as in the other two positions (non-degenerate). The base distribution in each codon position is significantly different from that in each other position. Position I has the most distinctive make up. Correlations demonstrate that in this ensemble of 1,265 codons positions I and II have the same type of composition as in the decoded mRNA for the average protein.

Base Sequence↗
PubMed1985

Functional analysis of lac repressor restart sites in translational initiation and reinitiation.

To define some of the features that influence ribosomal recognition of translational restart sites in the lac repressor mRNA, recombinant DNA methods have been used to construct lacI-Z fusions in which lacZ gene expression is dependent upon initiation or reinitiation within lacI mRNA sequences. Reinitiation efficiencies, as assessed by beta-galactosidase levels in strains bearing such plasmids, appear to be determined by at least three features of the RNA between the termination codon and reinitiation codon: the presence of competing out-of-frame AUG or GUG triplets, the distance between termination and reinitiation points, and the extent to which restart sequences remain accessible to ribosomes. While some of the restart sites are used with substantial efficiency for reinitiation, they do not function detectably as primary initiators if placed at the 5' end of the lacZ mRNA. This finding concurs with our observation that relative to the wild-type initiator region, which is recovered in quantitative yield from in vitro initiation reactions, ribosome protection of the four restart sites occurs at more than 100-fold lower efficiencies. In part, the lack of initiation activity is rationalized by the striking potential these sequences have for forming stable secondary structures that sequester elements essential for ribosome binding. However, the differential functioning of the restart sites in primary initiation versus reinitiation must also reflect real differences in the mechanisms operative in the two events.

Base Sequence↗
PubMed2000

Genomic structure and chromosomal mapping of the mouse hic-5 gene that encodes a focal adhesion protein.

The hic-5 gene encodes a focal adhesion protein that has striking similarity to paxillin. Genomic clones of the mouse hic-5 gene were isolated, and included 10 exons that covered the whole mouse mRNA sequence. Comparison of the sequence with those in the expressed sequence tag database suggested that the hic-5 gene contained an extra exon (named exon 1') located about 1kb upstream of exon 1, and mouse cells seemed to express two alternatively spliced forms of mRNA. All the exon-intron boundaries followed the GT/AG rule. Physical mapping and fluorescent in situ hybridization analysis indicated that the hic-5 gene is located on mouse chromosome 7, 60. 0cM from the centromere.

Amino Acid Sequence↗
PubMed1989

Hybridization analysis of RNA transported from rat liver nuclei in response to 35 kDa normal and 60 kDa messenger RNA transport factors.

The transport of messenger RNA (mRNA) in response to normal adult (35 kDa) and oncofetal (60 kDa) transport factors has been studied in a reconstituted cell-free system. Poly(A)+ mRNA sequences transported by the 35 kDa and 60 kDa transport factors were compared by cDNA:RNA hybridization kinetics. Heterologous hybridization reactions indicated that a proportion of messengers transported in response to the 35 kDa factor were absent or at a markedly reduced abundance in the mRNA released by the 60 kDa factor. Recombinant DNA probes containing cDNA inserts were used to quantitate transport of rat-liver-specific alpha 2 mu-globulin and albumin mRNA from isolated nuclei in presence of the normal and tumor-specific transport factors. More alpha 2 mu-globulin and albumin messenger sequences were transported in response to the 35 kDa transport factor as compared to the 60 kDa factor. These results indicate that the 35 kDa transport protein isolated from rat liver cytosol and the 60 kDa transport protein isolated from hepatoma cytosol, differ significantly in specificity for the classes of RNA sequences released from nuclei. Monoclonal antibodies against the 60 kDa factor do not cross-react with the 35 kDa factor or other proteins as determined by the immunobioassay and by the Western blot technique.

Albumins↗
PubMed1985

Displacement synthesis of globin complementary DNA: evidence for sequence amplification.

We have examined a cDNA displacement synthesis procedure in which the extent of precursor incorporation and the unusual kinetics of displacement synthesis suggest a unique replicative form of DNA and the occurrence of multiple rounds of displacement synthesis, leading to amplification of mRNA sequences. Globin double-stranded DNA containing a hairpin loop was extended by the addition of a homopolymer to the 3' end. This was followed by displacement synthesis with the Klenow fragment of DNA polymerase I that was primed by an oligonucleotide hybridized to the homopolymer. Thus, the hairpin cDNA was copied to form an open duplex with an inverted repetition of globin sequences. These molecules can then serve as templates for additional synthesis which would be primed from oligomers bound the homopolymer. Globin cDNA sequences appear to be amplified 10-fold or more by this procedure. Globin cDNA obtained by displacement synthesis was similar in size to the original template. However, displaced molecules associate to the extent that they are not readily resolved by electrophoresis or sedimentation under nondenaturing conditions. Restriction endonuclease digests of 32P-labeled displaced strands gave fragment patterns similar to rabbit globin cDNA hairpin molecules. S1 nuclease studies demonstrated that displaced complexes and replication intermediates are partially single stranded, which might account for their aggregation properties.

Animals↗
PubMed2000

Molecular characterization of a Siglec8 variant containing cytoplasmic tyrosine-based motifs, and mapping of the Siglec8 gene.

Through efforts to investigate the CD33-like subgroup of sialic acid binding immunoglobulin-like lectins (Siglecs), which are believed to be located on chromosome 19q13.4, we have identified the precise genomic region containing the Siglec8 gene. It is located on chromosome 19q13.4, approximately 330 kb downstream of the Siglec9 gene. Further, we have identified a novel Siglec8 variant, named Siglec8-Long (Siglec8-L), which differs in its last two exons from the previously published mRNA sequence of Siglec8 (GenBank Accession No. AF195092). Both Siglec8 and Siglec8-L are comprised of seven exons, of which the first five are identical, followed by marked differences in exon usage and mRNA splicing. The 499 amino acid protein encoded by the Siglec8-L open reading frame has a molecular weight of 54 kDa. Like the other members of the CD33-like subgroup of Siglecs, except for the previously published Siglec8, Siglec8-L also contains the two tyrosine-based motifs that have been found to recruit both SH2 domain-containing tyrosine and inositol phosphatases.

Alternative Splicing↗
PubMed1987

S4-alpha mRNA translation repression complex. I. Thermodynamics of formation.

Expression of the four ribosomal proteins from the Escherichia coli alpha operon (S4, S11, S13, and L17) is regulated at the level of translation by the binding of S4 to the alpha mRNA. Using a filter binding assay and alpha mRNA sequences prepared by in-vitro transcription, previous work located the S4 target site within the approximately 100-base leader sequence. We have extended this work to include fragments of the alpha leader with six different 5' end points and four different 3' end points. A core region between bases 23 and 69 (numbering from the first nucleotide of the E. coli transcript) binds S4 with an affinity of approximately 2 microM-1. Regions of weak interactions are located in the 22 nucleotides 5' and the 70 nucleotides 3' to this core; they increase the S4 affinity to approximately 13 microM-1. Studies of S4-alpha mRNA binding under different conditions have revealed the following. (1) Specific and non-specific binding show the same dependence on K+ concentration, with delta log+ K/delta log [K+] approximately 4 in most potassium salts. With KCl and KBr, much weaker salt dependence of specific complex formation is observed suggesting that the protein responds to the correct RNA substrate by binding halide anions. (2) Increasing the MgCl2 concentration between 1 and 4 mM enhances binding by a factor of 4, with no further effects up to 20 mM. About five Mg2+ are taken up by the complex with an average binding constant of approximately 600 M-1 each. Renaturation of the RNA in the presence of MgCl2 is also required to obtain full binding. These effects are seen only with alpha mRNA extending beyond the initiation codon; S4 binding to the alpha leader sequence itself is insensitive to Mg2+. (3) The association kinetics are fast and probably diffusion controlled. (4) Formation of the complex is entirely entropy driven.

Base Sequence↗