Rat aldolase A messenger RNA: the nucleotide sequence and multiple mRNA species with different 5'-terminal regions

The nucleotide sequence of aldolase A mRNA in rat skeletal muscle was determined using recombinant cDNA clones and a cDNA synthesized by primer extension. The sequence is composed of 1343 nucleotides (nt) except for the poly(A) tail. Based on the sequence analysis we have deduced an open reading frame with 363 amino acids (aa) (Mr 39134). The sequence suggests several nt polymorphisms in the mRNA population, one of which causes an aa change. The determined sequence of rat aldolase A mRNA was compared with the published ones of rabbit aldolase A or rat aldolase B mRNAs. The homology between rat and rabbit aldolase A mRNA sequences is greater than that between rat aldolase A and B mRNA sequences. Multiple aldolase A mRNAs having different Mrs were detected in the various tissues, and appeared to be expressed in a tissue-specific manner. Further analysis suggests that differences in mRNA length are due to differences in the 5'-noncoding terminal region.     

cDNA clone and expression analysis of rodent fast and slow skeletal muscle troponin I mRNAs

We have characterized the structure and expression of rodent mRNAs encoding the fast and slow skeletal muscle isoforms of the contractile regulatory protein, troponin I (TnIfast and TnIslow). TnIfast and TnIslow cDNA clones were isolated from mouse and rat muscle cDNA clone libraries and were used as isoform-specific probes in Northern blot and in situ hybridization studies. These studies showed that the TnIfast and TnIslow mRNAs are expressed in skeletal muscle, but not cardiac muscle or other tissues, and that they are differentially expressed in individual muscle fibers. Fiber typing on the basis of in situ hybridization analysis of TnI isoform mRNA content showed an excellent correlation with fiber type as assessed by myosin ATPase histochemistry. These results directly demonstrate that the differential expression of skeletal muscle TnI isoforms in the various classes of vertebrate striated muscle cells is based on gene regulatory mechanisms which control the abundances of specific TnI mRNAs in individual muscle cells. Both TnIfast and TnIslow mRNAs are expressed, at comparable levels, in differentiated cultures of rat L6 and mouse C2 muscle cell lines. Thus, although neuronal input has been shown to be an important factor in determining fast versus slow isoform-specific expression in skeletal muscle, both TnIfast and TnIslow genes can be expressed in muscle cells in the absence of nerve. Comparison of the deduced rodent TnI amino acid sequences with previously determined rabbit protein sequences showed that residues with potential fast/slow isoform-specific function are present in several discrete clusters, two of which are located near previously identified actin and troponin C binding sites.     

Differential control of tropomyosin mRNA levels during myogenesis suggests the existence of an isoform competition-autoregulatory compensation control mechanism.

We have isolated tropomyosin cDNAs from human skeletal muscle and nonmuscle cDNA libraries and constructed gene-specific DNA probes for each of the four functional tropomyosin genes. These DNA probes were used to define the regulation of the corresponding mRNAs during the process of myogenesis. Tropomyosin regulation was compared with that of beta- and gamma-actin. No two striated muscle-specific tropomyosin mRNAs are coordinately accumulated during myogenesis nor in adult striated muscles. Similarly, no two nonmuscle tropomyosins are coordinately repressed during myogenesis. However, mRNAs encoding the 248 amino acid nonmuscle tropomyosins and beta- and gamma-actin are more persistent in adult skeletal muscle than those encoding the 284 amino acid nonmuscle tropomyosins. In particular, the nonmuscle tropomyosin Tm4 is expressed at similar levels in adult rat nonmuscle and striated muscle tissues. We conclude that each tropomyosin mRNA has its own unique determinants of accumulation and that the 248 amino acid nonmuscle tropomyosins may have a role in the architecture of the adult myofiber. The variable regulation of nonmuscle isoforms during myogenesis suggests that the different isoforms compete for inclusion into cellular structures and that compensating autoregulation of mRNA levels bring gene expression into alignment with the competitiveness of each individual gene product. Such an isoform competition-autoregulatory compensation mechanism would readily explain the unique regulation of each gene.     

Selective gene expression during sporulation of Physarum polycephalum.

The two-dimensional gel electrophoresis of polypeptides synthesized in vitro from poly(A)+ RNA showed that mRNA populations change during sporulation of Physarum polycephalum. The differential hybridization of a cDNA library prepared from poly(A)+ RNA isolated from sporulating cells revealed that of 846 clones, 64 corresponded to sporulation-specific mRNAs. Further analysis demonstrated that these clones contained seven different sequences: three abundant sequences composing 3.2, 1.8, and 1.2% of the library and four other less abundant sequences. It is probable that all the major mRNAs specifically expressed in early stages of sporulation were identified. The most abundant mRNA from this group coded for a hydrophobic protein that contained a signal peptide. This protein is 47% similar to another Physarum protein, which was encoded by the most abundant plasmodium-specific mRNA. The plasmodial mRNA was degraded during sporulation and was replaced by the sporulation mRNA. These two proteins are thus encoded by members of a gene family whose expression is developmentally regulated.     

Characterization of cDNA coding for the complete light meromyosin portion of a rabbit fast skeletal muscle myosin heavy chain

Myosin-heavy-chain-specific cDNA clones have been isolated from a cDNA library prepared from hind leg muscle of a 14-day-old rabbit. According to restriction enzyme analysis these can be grouped into at least two, probably three different classes. RNA dot-blot hybridization shows that all of these clones correspond to mRNAs expressed in fast skeletal muscle. The clones of the most abundant form, class I, can be aligned to cover the complete light meromyosin portion of myosin heavy chain. The sequence of the coding and the 3'-untranslated region, together comprising 2143 base pairs, has been determined. The class I clone detects a multigene family of 8-12 members on a Southern blot of rabbit genomic DNA.     

Cloning and regulation of rat apolipoprotein B mRNA

Recombinant cDNA clones that code for apolipoprotein B(apoB) were isolated from a rat liver cDNA library, using synthetic oligonucleotide probe derived from the sequence of human apoB cDNA. The nucleotide and deduced amino acid sequences of the rat apoB clone pRB5, 1.2 kb in length, showed 83% and 84% homology to those of human apoB. Northern blot analysis revealed that rat apoB cDNA probe cross-reacts with human and rabbit apoB mRNA sequences and the size of those mRNAs, approximately 15 kb long, were not discernibly different. In addition, apoB mRNA was abundant only in the liver and intestine. Finally, cholesterol feeding to rats for six weeks resulted in a several-fold increase in the level of apoB mRNA in the liver.     

Molecular cloning and characterization of cDNA for androgen-repressible rat liver protein, SMP-2

SMP-2 is a rat liver protein whose synthesis is influenced by both androgens and aging. The steady-state level of its mRNA is repressed by the androgen. Compared to the adult male, SMP-2 mRNA is found in higher amounts in the prepubertal and senescent male rat livers which show relative androgen insensitivity. A cDNA library in the plasmid pBR322 was constructed from the female rat liver which contains a high level of SMP-2 mRNAs. Recombinant plasmids were screened by differential colony hybridization to 32P-labeled single-stranded cDNAs from adult female and adult male hepatic poly(A)+ RNAs. From a total of 3500 recombinant clones, 11 highly female specific clones were identified. From these female specific colonies the SMP-2 cDNA-containing plasmid (pSP11) was identified by its ability to select an mRNA species whose translation product is immunochemically and electrophoretically indistinguishable from SMP-2. This insert represents a 571-base pair portion of the SMP-2 cDNA. Rescreening of the library at a high colony density using the 32P-labeled cDNA insert of pSP11 identified several positive clones with larger inserts. Hybrid-selected mRNA translation again confirmed these clones to carry SMP-2 cDNA sequences. The plasmid pSP4a containing a 1040-base pair cDNA insert of SMP-2 was characterized by DNA sequence analysis. The size of the cDNA insert of pSP4a is close to the estimated size of the SMP-2 mRNA. The cDNA sequence provides an open reading frame of 282 amino acid residues. A comparison of the translated amino acid sequence with the protein sequences of NBRF-PIR, PSQNEW, and LOSALA data bases did not establish any sequence homology with known proteins. Northern blot analysis using the 32P-labeled cDNA insert of pSP4a confirms the androgenic repression of the SMP-2 mRNA.     

Molecular cloning of mRNAs expressed specifically during spherulation of Physarum polycephalum

A cDNA library was constructed using the poly(A)+ RNA extracted from spherulating Physarum polycephalum microplasmodia. This library (740 clones) was screened by differential hybridization with 32P-labeled poly(A)+ RNA from growing plasmodia and developing spherules. The results showed that at least 30% of the clones corresponded to mRNAs expressed specifically in spherulating plasmodia. The 35 spherulation-specific cDNA clones giving the strongest hybridization signals were analysed. From this group, four different sequences complementary to very abundant mRNAs were identified. They each accounted for 1.5% of 4.5% of all the clones in the library and probably represented the most abundant spherulation-specific mRNAs. In addition, four less abundant mRNAs were identified from stage-specific clones giving weaker hybridization signals. These sequences represented individually between 0.3% and 0.7% of the clones in the library. Northern blots showed that these eight different sequences were absent from plasmodia and were most abundant 24-36 h after the induction of spherulation. Similar results were also obtained when spherulation was induced by the addition of a sublethal concentration of ferrous iron ions to the growth medium. Hybridization of the spherule-specific clones to Southern blots of genomic DNA suggested the presence of one copy for each gene.     

A monoclonal antibody, 3A10, recognizes a specific amino acid sequence present on a series of developmentally expressed brain proteins

Immunoblotting showed that a monoclonal antibody, 3A10, binds to a series of rat brain-specific antigens with molecular masses of 150-, 120-, 118-, 106-, 104-, 79-, and 77-kDa. The expression of 3A10 antigens is dependent on the developmental stage of the brain; only the 106-kDa antigen is detected during embryonic stages of rat brain development, while the expression of the remaining 6 antigens starts after birth and reaches a maximum during postnatal days 15-21. Detection of the 3A10 antigens in cultured neuronal and glial cells derived from cerebral cortices of rat brain at embryonic day 18 showed that the 77-, 79-, 106-, and 150-kDa antigens are specifically expressed in neuronal cells. The 77-kDa antigen was purified and identified as synapsin I by amino acid sequence analyses of the peptide fragments isolated after Achromobacter protease I treatment. During the isolation of 3A10-reactive proteins by immunological screening of cDNA libraries constructed from adult rat brain, we found that all of the 3A10-reactive clones contain nucleotide sequences encoding the unique amino acid sequence TRSP(S, R,G)P. Analyses of 3A10-binding to various synthetic peptides showed that the monoclonal antibody recognizes a specific conformational structure formed by either the TRSPXP sequence or similar amino acid sequences that are expressed on a series of developmentally expressed brain proteins.     

Tubulin tyrosine ligase: Protein and mRNA expression in developing rat skeletal muscle

Alpha tubulin can be post-translationally tyrosinated at the carboxy-terminus by a specific enzyme: tubulin tyrosine ligase. The expression of tubulin tyrosine ligase mRNA and protein during the development of rat skeletal muscle was examined in the present study. A portion of the coding region of the rat ligase cDNA was isolated and sequenced. The nucleotide and amino acid sequences showed about 90% homology with previously reported porcine and bovine ligase sequences. In newborn rats, ligase mRNA and protein were highly expressed in skeletal muscle. During early postnatal development, however, both ligase mRNA and protein dropped down dramatically. Quantitative measurements revealed that ligase protein at postnatal day 20 represented only 10% or less of the level at postnatal day 1. Ligase mRNA expression was also examined during the myogenesis in vitro . A strong ligase mRNA signal was detected in both undifferentiated myoblasts and cross-striated, contractile myotubes. The present results suggest that, during muscle differentiation, ligase function may be regulated by the amount of available mRNA. The discrepancy in the ligase expression between the in vivo and in vitro myogenesis suggests that factors controlling the levels of mRNA in vivo are lost in vitro .     

The rat gene encoding neurotensin and neuromedin N. Structure, tissue-specific expression, and evolution of exon sequences

Recombinant DNA clones encoding the neurotensin/neuromedin N precursor protein have been isolated from both bovine hypothalamus cDNA and rat genomic libraries using a heterologous canine cDNA probe. Nucleotide sequence analysis of these clones and comparison with the previously determined canine sequence has revealed that 76% of the amino acid residues are conserved in all three species. The protein precursor sequences predicted from bovine hypothalamus and canine intestine cDNA clones vary at only 9 of 170 amino acid residues suggesting that within a species identical precursors are synthesized in both the central nervous system and intestine. The rat gene spans approximately 10.2 kilobases (kb) and is divided into four exons by three introns. The neurotensin and neuromedin N coding domains are tandemly positioned on exon 4. RNA blot analysis has revealed that the rat gene is transcribed to yield two distinct mRNAs, 1.0 and 1.5 kb in size, in all gastrointestinal and all neural tissues examined except the cerebellum. There is a striking variation in the relative levels of these two mRNAs between brain and intestine. The smaller 1.0-kb mRNA greatly predominates in intestine while both mRNA species are nearly equally abundant in hypothalamus, brain stem, and cortex. Sequence comparisons and RNA blot analysis indicate that these two mRNAs result from the differential utilization of two consensus poly(A) addition signals and differ in the extent of their 3' untranslated regions. The relative combined levels of the mRNAs in various brain and intestine regions correspond roughly with the relative levels of immunologically detectable neurotensin except in the cerebral cortex where mRNA levels are 6 times higher than anticipated.     

The full length coding sequence of rat liver androsterone UDP-glucuronyltransferase cDNA and comparison with other members of this gene family.

Cloned cDNAs coding for hepatic UDP-glucuronyltransferase (UDPGT) have been isolated from a rat liver cDNA library in the expression vector bacteriophage lambda gt11 using anti-UDPGT antibodies. Four different mRNAs have been identified by sequencing of 15 UDPGT cDNA clones. The sequences of the four classes of cDNA were determined to be 85-95% homologous. Restriction fragments were isolated from the cDNA in each class and used as class specific probes. Hybridisation of these probes to northern blots of total RNA prepared from the livers of normal and genetically deficient Wistar rats identified the cDNA in class 4 with androsterone UDPGT. Translation of the cDNA sequence of clone rlug 23, the longest member of class 4, allowed determination of the complete amino acid sequence of androsterone UDPGT.     

Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation.

The molecular basis for the expression of rat embryonic fibroblast tropomyosin 1 and skeletal muscle beta-tropomyosin was determined. cDNA clones encoding these tropomyosin isoforms exhibit complete identity except for two carboxy-proximal regions (amino acids 189 to 213 and 258 to 284) and different 3'-untranslated sequences. The isoform-specific regions delineate the troponin T-binding domains of skeletal muscle tropomyosin. Analysis of genomic clones indicates that there are two separate loci in the rat genome that contain sequences complementary to these mRNAs. One locus is a pseudogene. The other locus contains a single gene made up of 11 exons and spans approximately 10 kilobases. Sequences common to all mRNAs were found in exons 1 through 5 (amino acids 1 to 188) and exons 8 and 9 (amino acids 214 to 257). Exons 6 and 11 are specific for fibroblast mRNA (amino acids 189 to 213 and 258 to 284, respectively), while exons 7 and 10 are specific for skeletal muscle mRNA (amino acids 189 to 213 and 258 to 284, respectively). In addition, exons 10 and 11 each contain the entire 3'-untranslated sequences of the respective mRNAs including the polyadenylation site. Although the gene is also expressed in smooth muscle (stomach, uterus, and vas deferens), only the fibroblast-type splice products can be detected in these tissues. S1 and primer extension analyses indicate that all mRNAs expressed from this gene are transcribed from a single promoter. The promoter was found to contain G-C-rich sequences, a TATA-like sequence TTTTA, no identifiable CCAAT box, and two putative Sp1-binding sites.     

Generation of troponin T isoforms by alternative RNA splicing in avian skeletal muscle. Conserved and divergent features in birds and mammals

We describe the isolation and sequence analysis of quail muscle cDNA clones encoding two closely related isoforms of the striated muscle contractile protein, troponin T. The cDNAs represent two troponin T mRNAs that exhibit an unusual sequence relationship. The two mRNAs have identical sequences over hundreds of nucleotides including 3' untranslated regions, but they differ dramatically in a discrete, internally located block of 38 nucleotides. The two alternative sequences of this 38-nucleotide block encode two different but related versions of amino acid residues 230-242, near the C terminus of the protein. These results are consistent with a novel mechanism of troponin T isoform generation by alternative mRNA splicing pathways from a single gene containing two different exons corresponding to amino acids 229-242, as recently proposed by Medford et al. (Medford, R. M., Nguyen, H. T., Destree, A. T., Summers, E., and Nadal-Ginard, B. (1984) Cell 38, 409-421). This proposal was based on analysis of a rat troponin T genomic DNA clone and a cDNA clone corresponding to one of the two alternatively spliced mRNAs. Our analysis of quail troponin T cDNA clones, apparently corresponding to two alternatively spliced mRNA species, provides important new evidence for this novel mechanism of troponin T isoform generation and reveals the differential splicing mechanism to be of great antiquity, antedating the bird-mammal divergence. One of the quail alternative isoform sequences clearly corresponds to one of the rat sequences, but the other quail alternative sequence does not correspond to either of the rat sequences. This result suggests a greater complexity of troponin T gene structure or a greater diversity of troponin T isoform genes than is currently known, and also has implications for the functional significance of the troponin T protein isoform heterogeneity. Comparison of quail and mammal alternative isoform sequences also reveals strongly conserved features which suggest that all the isoform alternative amino acid sequences are variations on a common structural theme.     

In vivo developmental modifications of the expression of genes encoding muscle-specific enzymes in rat

cDNA clones for rat muscle-type creatine kinase and glycogen phosphorylase and aldolase A were isolated from a rat muscle cDNA library. An additional clone recognizing an unidentified 2.7-kilobase pair mRNA species was also isolated. These cDNA clones were used as probes to investigate the expression of the corresponding mRNAs during muscle development. Two aldolase A mRNA species were detected, one of 1650 bases expressed in non-muscle tissues, fetal muscle, and adult slow-twitch muscle, the other of 1550 bases was highly specific of adult fast-twitch skeletal muscle differentiation. These aldolase A mRNAs were shown by primer extension to differ by their 5' ends. The accumulation of muscle-type phosphorylase and creatine kinase and muscle-specific aldolase A mRNA accumulation during muscle development seems to be a coordinate process occurring progressively from the 17th day of intrauterine life up to the 30th day after birth. In contrast, the 2.7-kilobase pair RNA species is maximally expressed at the 1st week after birth as is the neonatal form of myosin heavy chain mRNA.     

Atlantic salmon (Salmo salar) serum albumin: cDNA sequence, evolution, and tissue expression

Atlantic salmon serum albumin is one of the most abundant proteins in salmon liver, representing 1.6% of all clones in a cDNA library made from salmon liver RNA. The DNA from a number of clones was sequenced to reveal an open reading frame of 1,827 bases encoding a 608-amino-acid protein. The sequenced 5' untranslated region is 69 bases long and the 3' untranslated region contains two putative polyadenylation signals and poly(A) tail. Sequence analysis of different clones indicates the presence of a second cDNA for salmon serum albumin. Multiple alignments of salmon serum albumin deduced amino acid sequence with Xenopus laevis, rat, bovine, and human serum albumins shows significant conservation of cysteine residues. The triple domain structure of serum albumin proteins is maintained. Unlike mammalian systems where serum albumin expression appears to be specific to liver only, salmon serum albumin is expressed in muscle also.