Alternative splicing of the human isoaspartyl protein carboxyl methyltransferase RNA leads to the generation of a C-terminal -RDEL sequence in isozyme II.

We have isolated two cDNA clones that correspond to the mRNAs for two isozymes of the human L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77). The DNA sequence of one of these encodes the amino acid sequence of the C-terminal half of the human erythrocyte isozyme I. The other cDNA clone includes the complete coding region of the more acidic isozyme II. With the exception of potential polymorphic sites at amino acid residues 119 and 205, the deduced amino acid sequences differ only at the C-terminus, where the -RWK sequence of isozyme I is replaced by a -RDEL sequence in isozyme II. The latter sequence is identical to a mammalian endoplasmic reticulum retention signal. With the previous evidence for only a single gene for the L-isoaspartyl/D-aspartyl methyltransferase in humans, and with evidence for consensus sites for alternative splicing in corresponding mouse genomic clones, we suggest that alternative splicing reactions can generate the major isozymes previously identified in human erythrocytes. The presence of alternative splicing leads us to predict the existence of a third isozyme with a -R C-terminus. The calculated isoelectric point of this third form is similar to that of a previously detected but uncharacterized minor methyltransferase activity.     

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.     

Human cellular retinol-binding protein gene organization and chromosomal location

The gene encoding the human cellular retinol-binding protein (CRBP) has been isolated from genomic libraries and its structure determined. Only one copy of the gene is present in the human genome. We have located the CRBP gene to segment 3p11-3qter on human chromosome 3 using hybridizations to mouse-human, rat-human and hamster-human cell hybrids. The gene harbors four exons encoding 24, 59, 33, and 16 amino acid residues respectively. The second intervening sequence alone occupies 19 kb of the 21 kb of the CRBP gene. The nucleotide sequence of the gene has been determined with the exception of the second intron. The positions of the introns agree with those in the rat CRBPII, the rat liver fatty-acid-binding protein and the mouse adipose P2 protein genes encoding molecules belonging to the same protein family as CRBP. In contrast to the other sequenced members of this family the promoter of the CRBP gene resembles those found in the 'housekeeping' genes in that it is (G + C)-rich, contains multiple copies of the CCGCCC sequence and lacks TATA box. A 9-bp homology containing the core sequence of the simian virus 40 enhancer repeat was found in the 5' upstream region. A genomic Southern blot probed with CRBP cDNA revealed hybridizing bands in restricted chicken and frog DNA.     

Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

Characterization of the gene for human plasminogen, a key proenzyme in the fibrinolytic system

The organization and structure of the gene coding for plasminogen has been determined by a combination of in vitro amplification of leukocyte DNA from normal individuals and isolation of unique clones from three different human genomic libraries. These clones were characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene for human plasminogen spanned about 52.5 kilobases of DNA and consisted of 19 exons separated by 18 introns. DNA sequence analysis revealed that the five kringle structures in plasminogen were coded by two exons. The nucleotides in the introns at the intron-exon boundaries were GT-AG analogous to those found in other eukaryotic genes. Three polyadenylation sites for plasminogen mRNA were also identified. When the amino acid sequences deduced from the genomic DNA and cDNAs of plasminogen were compared with that of the plasma protein determined by amino acid sequence analysis, an apparent amino acid polymorphism was observed in several positions of the polypeptide chain. Nucleotide sequence analysis of the amplified genomic DNAs and genomic clones also revealed that the plasminogen gene was very closely related to several other proteins, including apolipoprotein(a). This protein may have evolved via duplication and exon shuffling of the plasminogen gene. The presence of another plasminogen-related gene(s) in the human genomic library was also observed.     

The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing

The complete nucleotide sequences of rat M1- and M2-type pyruvate kinase mRNAs were determined by sequencing the cDNAs and by analyses of S1 nuclease mapping and primer extension. The sequences have an identical molecular size of about 2220 nucleotides excluding a poly(A) tail and include 1593-nucleotide coding region. Their nucleotide sequences are identical except for 160-nucleotide sequences within the coding regions. The amino acid sequences of the M1- and M2-type subunits deduced from the cDNA sequences differ by only 45 residues within domain C, which constitutes the main region responsible for intersubunit contact. The sequence of this region of the M2-type shows higher homology than that of the M1-type with the corresponding sequence of the L-type. Since the M2- and L-types are allosteric enzymes, unlike to the M1-type, the residues common to the M2- and L-types, but not the M1-type may be important for mediating the allosteric properties. Genomic clones encoding both M1- and M2-type isozyme mRNAs were isolated. By partial sequence analysis of a clone lambda MPK37 four exons were identified, of which two adjacent exons coded the M1- and M2-specific sequences, respectively. The two remaining exons present downstream coded amino acids common to the two isozymes. Thus, we conclude that the M1- and M2-type isozymes of pyruvate kinase are produced from the same gene probably by alternative RNA splicing.     

Molecular cloning and functional expression of two splice forms of human N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase.

We have isolated and sequenced human cDNA and mouse genomic DNA clones encoding N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (phosphodiester alpha-GlcNAcase) which catalyzes the second step in the synthesis of the mannose 6-phosphate recognition signal on lysosomal enzymes. The gene is organized into 10 exons. The protein sequence encoded by the clones shows 80% identity between human and mouse phosphodiester alpha-GlcNAcase and no homology to other known proteins. It predicts a type I membrane-spanning glycoprotein of 514 amino acids containing a 24-amino acid signal sequence, a luminal domain of 422 residues with six potential N-linked glycosylation sites, a single 27-residue transmembrane region, and a 41-residue cytoplasmic tail that contains both a tyrosine-based and an NPF internalization motif. Human brain expressed sequence tags lack a 102-base pair region present in human liver cDNA that corresponds to exon 8 in the genomic DNA and probably arises via alternative splicing. COS cells transfected with the human cDNA expressed 50-100-fold increases in phosphodiester alpha-GlcNAcase activity proving that the cDNA encodes the subunits of the tetrameric enzyme. Transfection with cDNA lacking the 102-base pair region also gave active enzyme. The complete genomic sequence of human phosphodiester alpha-GlcNAcase was recently deposited in the data base. It showed that our cDNA clone was missing only the 5'-untranslated region and initiator methionine and revealed that the human genomic DNA has the same exon organization as the mouse gene.     

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.     

Nucleotide sequence of the gene for human prothrombin

A human genomic DNA library was screened for the gene coding for human prothrombin with a cDNA coding for the human protein. Eighty-one positive lambda phage were identified, and three were chosen for further characterization. These three phage hybridized with 5' and/or 3' probes prepared from the prothrombin cDNA. The complete DNA sequence of 21 kilobases of the human prothrombin gene was determined and included a 4.9-kilobase region that was previously sequenced. The gene for human prothrombin contains 14 exons separated by 13 intervening sequences. The exons range in size from 25 to 315 base pairs, while the introns range from 84 to 9447 base pairs. Ninety percent of the gene is composed of intervening sequence. All the intron splice junctions are consistent with sequences found in other eukaryotic genes, except for the presence of GC rather than GT on the 5' end of intervening sequence L. Thirty copies of Alu repetitive DNA and two copies of partial KpnI repeats were identified in clusters within several of the intervening sequences, and these repeats represent 40% of the DNA sequence of the gene. The size, distribution, and sequence homology of the introns within the gene were then compared to those of the genes for the other vitamin K dependent proteins and several other serine proteases.     

Structure of the horseradish peroxidase isozyme C genes

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.