Expression of three mRNA species from a single rat aldolase A gene, differing in their 5' non-coding regions

The complete nucleotide sequence of the rat aldolase A isozyme gene, including the 5' and 3' flanking sequences, was determined. The gene comprises ten exons, spans 4827 base-pairs and occurs in a single copy per haploid rat genome. The genomic DNA sequence was compared with those of three species of rat aldolase A mRNA (mRNAs I, II and III) that have been found to differ from each other only in the 5' non-coding region and to be expressed tissue-specifically. It revealed that the first exon (exon M1) encodes the 5' non-coding sequence of mRNA I, while the second exon (exon AH1) encodes those of mRNAs II and III and the following eight exons (exons 2 to 9) are shared commonly by all the mRNA species. These results allowed us to conclude that mRNA I and mRNAs II, III were generated from a single aldolase A gene by alternative usage of exon M1 or exon AH1 in addition to exons 2 to 9. S1 nuclease mapping of the 5' ends of their precursor RNAs suggested that these three mRNA species were transcribed from three different initiation sites on the single gene.     

Nucleotide sequence of a cDNA clone for human aldolase: a messenger RNA in the liver

Nearly complete cDNA clones for human aldolase A mRNA were isolated from human liver cDNA library and the nucleotide sequence determined. Using the cDNA clone as a probe the length of human aldolase A mRNAs, isolated from the skeletal muscle, liver and placenta tissues, was measured by RNA blotting and estimated to be 1,600 nucleotides for skeletal muscle mRNA and 1,700 nucleotides for both the liver and placenta mRNAs, indicating that different species of mRNA coding for human aldolase A were expressed in the different tissues.     

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.     

Complete sequence of ovine alpha s2-casein messenger RNA

The primary structure of mRNA coding for ovine alpha s2 casein has been determined by chemical sequencing of three cDNA clones and the primer extension products of the longest one. The mRNA was 1,024 nucleotides long, excluding the poly(A) tail. The length of the 5' noncoding, coding and 3' noncoding regions was 53, 669 and 302 nucleotides, respectively. A comparison of the nucleotide sequence of ovine alpha s2-casein and guinea-pig casein A mRNAs revealed an extensive homology in the 5' and 3' noncoding regions. The deduced amino acid sequence of ovine alpha s2-casein was compared with its bovine and guinea-pig counterparts. Moreover, an heterogeneity was evidenced in the mRNA population of the alpha s2-casein.     

Discrimination among multiple AATAAA sequences correlates with interspecies conservation of select 3'' untranslated nucleotides.

The DNA sequence corresponding to the 1.3 kb 3' untranslated region of the 6.5 kb human procollagen alpha 1(IV) mRNA was determined and compared with the mouse sequence obtained from 3' cDNA and genomic clones overlapping the reported 5' half (Oberbaumer et al., 1985, Eur. J. Biochem. 147:217). Although four AAUAAA hexanucleotides are found in the human and seven in the mouse RNAs, Northern blot hybridization showed almost exclusive utilization of the most 3' sequence, in contrast to the pattern seen when using alpha 1(I), alpha 2(I), alpha 1(III) and alpha 2(V) procollagen probes. Moreover, the ninety nucleotides 5' to the poly A tail in the major alpha 1(IV) mRNAs exhibit a much greater degree of interspecies homology than those encompassing the other three shared AAUAAA recognition signals. Further examination of this highly conserved area revealed the presence of two "consensus sequences" found in the 3' noncoding region of a number of RNA polymerase II transcribed genes (Mattaj and Zeller, 1983, Embo J. 2:1883) and, unexpectedly, some similarity with the nucleotides 5' to the poly A attachment signals in other procollagen mRNAs.     

Complete amino acid sequence of the type II isozyme of rat hexokinase, deduced from the cloned cDNA: comparison with a hexokinase from novikoff ascites tumor

The 917-residue amino acid sequence of the Type II isozyme of rat hexokinase has been deduced from the nucleotide sequence of cloned cDNA. The sequences of 197 nucleotides in the 5' untranslated region and 687 bases of the 3' untranslated region have also been determined. A region of overlap between two discrete cDNA clones was confirmed by isolation and sequencing of a genomic DNA clone that spanned the region. Within this region, the 634-nucleotide coding sequence was divided into three exons, each of 150-250 nucleotides; these results suggest that the gene encoding Type II hexokinase is likely to be quite complex. There is extensive similarity between the sequences of the N- and C-terminal halves of the Type II isozyme, as previously seen with the Type I and III isozymes; this is consistent with the view that these enzymes evolved by a process of gene duplication and fusion. A cDNA encoding the entire C-terminal half of a hexokinase from Novikoff ascites tumor cells was also isolated and found to be identical to a cDNA encoding the corresponding region of the Type II isozyme of skeletal muscle. Northern analysis indicated that a single mRNA, approx 5200 nucleotides in length, encoded both the skeletal muscle and the tumor enzymes. These results do not support previous speculation that the hexokinase isozymes of normal tissue are distinct from those of tumors, and suggest the possibility that post-translational modifications of a single protein species might account for apparent differences between the isozymes of normal and tumor tissues.     

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.     

The nucleotide sequence and derived amino acid sequence of cDNA coding for mouse carbonic anhydrase II

The nucleotide sequence of a clone containing mouse carbonic anhydrase (CA) cDNA in pBR322 has been determined. The cloned cDNA contains all of the coding region except for nucleotides specifying the first eight amino acids, and all of the 3' noncoding region, which consists of 700 nucleotides. A cDNA clone was identified which contains an additional 54 bp at the 5' end, so that the complete amino acid sequence of mouse CA could be deduced. This sequence showed a 73-81% homology with other mammalian CA form II isozymes, 56-63% with form I isozymes, and 52-56% with form III isozymes. By examination of the amino acids which are unique and invariant for each isozyme, the mouse amino acid sequence was found to contain 16 of the 23 residues that are unique and invariant to mammalian CA form II isozymes, but only one or no residue for forms I and III, respectively.     

Expression of calpastatin isoforms in muscle and functionality of multiple calpastatin promoters

Calpastatin is the specific endogenous inhibitor of calpain proteinase that is encoded by a single gene. Transient transfection assays in both a non-fusing skeletal muscle and non-muscle cell-line demonstrated that the putative porcine calpastatin promoter regions 5' to exons 1xa, 1xb, and 1u were functional. Both real-time quantitative and semi-quantitative RT-PCR on porcine skeletal muscle total RNA indicated that steady-state expression of Type I and III mRNAs containing exons 1xa and 1u, respectively, was at equivalent levels whilst the expression of Type II mRNA containing exon 1xb was significantly less (p<0.001). Immunoprobing of Western blotted muscle extracts with an antibody raised against a peptide sequence encoded by exon 1xa indicated that Type I protein was expressed and that there was significantly more Type I protein in cardiac than skeletal muscle (p<0.001). The results suggest that the expression of the single calpastatin gene was differentially controlled at several levels.     

Isolation and nucleotide sequence of a full-length cDNA coding for aldolase B from human liver.

Two recombinant clones, pA2 and pA3, containing cDNA sequences for human aldolase B have been isolated from a full length human liver cDNA library. The larger one, pA3, has been subcloned in M13 phage and completely sequenced with the chain terminator method. The sequence covers 1,600 nucleotides including the whole coding region (1,050 nucleotides), 67 nucleotides from the 5' non-coding region and the whole 3' non-coding region, 440 nucleotides long, down to the poly-A tail. Comparison with rabbit aldolase A and with a partial sequence of rat aldolase B, shows a homology of about 76% for aldolase A and of about 94% for aldolase B, which indicates that the sequenced cDNA codes for the liver isoenzyme. This is the first complete sequence reported for human aldolase B. The pA3 clone strongly hybridizes to 18S mRNA from human adult liver as expected from the size of the isolated cDNA.     

Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5'' noncoding region.

Poliovirus mRNA contains a long 5' noncoding region of about 750 nucleotides (the exact number varies among the three virus serotypes), which contains several AUG codons upstream of the major initiator AUG. Unlike most eucaryotic mRNAs, poliovirus does not contain a m7GpppX (where X is any nucleotide) cap structure at its 5' end and is translated by a cap-independent mechanism. To study the manner by which poliovirus mRNA is expressed, we examined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. In this paper we report striking translation system-specific differences in the ability of the altered mRNAs to be translated. The results suggest the existence of an inhibitory cis-acting element(s) within the 5' noncoding region of poliovirus (between nucleotides 70 and 381) which restricts mRNA translation in reticulocyte lysate, wheat germ extract, and Xenopus oocytes, but not in HeLa cell extracts. In addition, we show that HeLa cell extracts contain a trans-acting factor(s) that overcomes this restriction.     

Predicted structures of apolipoprotein II mRNA constrained by nuclease and dimethyl sulfate reactivity: stable secondary structures occur predominantly in local domains via intraexonic base pairing

Analyses of apolipoprotein II mRNA with chemical and enzymatic probes showed that double- and single-stranded regions were distributed uniformly along the mRNA except for a large (72 nucleotides) single-stranded region containing the translation stop codon. Secondary structure models constrained by the experimental data were made by varying the distance (along the mRNA) over which base pairing was allowed. Four prominent secondary structures were seen with restrictions of 165, 330, or 659 nucleotides suggesting that such structures from via local interactions over distances of 50-120 nucleotides. Predicted long range interactions involve only 2-3 base pairs while local interactions involve helices of 4-10 base pairs. Predicted helices of greater than or equal to 4 base pairs occur primarily within exons, raising the possibility that prominent secondary structures in mRNAs may be largely due to intraexonic base pairing. Tests of single- and double-stranded domains by oligonucleotide-directed RNase H cleavage and primer extension were in accord with the structure model and with nuclease and chemical modification data. The model predicting base pairing between the coding and the 3' noncoding regions was tested by RNase H cleavage followed by oligo(dT)-cellulose chromatography to separate 5' and 3' mRNA fragments. Most (82%) of the 5' fragment remained associated with the 3' noncoding region in a structure with a tm = 50 degrees C in 0.2 M Na+ suggesting that this stem could be stable in vivo. This stem may be stable in the isolated mRNA, but would likely occur transiently in polyribosomal apolipoprotein II mRNA due to ribosome transit through the 5' side of the stem. Alternate structures may occur in this region during ribosome transit and play a role in translation termination or in determining the susceptibility of the mRNA to degradation.