Sequence analysis of two exons from the murine dystrophin locus

We have isolated two genomic clones from the murine dystrophin locus, containing single exons encoding protein sequence from the putative actin-binding domain of the amino-terminus and the terminal portion of the triple helical domain. Using interspecific backcross progeny mice, both clones were shown to be X-linked. Sequence analysis indicated that the amino-terminal clone contains a 173 bp exon exhibiting 90% nucleotide sequence identity to human dystrophin exon 6, whilst the C-terminal clone contains a 61 bp exon with 93% nucleotide sequence identity to the human cDNA sequence.The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the accession numbers X58153 and X57745.     

Characterization of the CYP4A11 gene,a second CYP4A gene in humans

Comparison between the cDNA sequence of CYP4A11 and that deduced from a published genomic clone suggested the presence of an additional CYP4A gene in humans, CYP4A22. PCR amplification of genomic DNA yielded overlapping clones covering 13kb of genomic DNA and extending from 1003bp upstream from CYP4A11 translation initiation to 135bp upstream of the mRNA polyadenylation signal. Sequence and Southern blot analysis showed the presence in humans of two highly homologous CYP4A genes, CYP4A11 and CYP4A22. These two genes share 96% sequence identity and have similar intron/exon sizes and distribution. Short nucleotide insertions (< or =10bp) in introns 1, 3, 9, and 11, and deletions (< or =18bp) in introns 4, 6, and 11 differentiate the two genes. RT-PCR amplification of human kidney RNA followed by restriction fragment analysis showed that CYP4A11 is the predominant isoform expressed in kidney.     

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

Genetics and expression of two pectinesterase genes in Valencia orange

The genetics and expression of pectinesterase (PE) genes were examined in Valencia orange. Degenerate primers based on partial amino acid sequence of a 36 kDa PE protein isolated from juice vesicles were used to amplify a 350 bp DNA fragment from cDNA prepared from juice vesicle total RNA. Two groups of 350 bp PE clones with 66% sequence identity were isolated. A clone from each group was used to screen a Valencia orange genomic DNA λ library. Two different lambda clones that contained complete PE coding sequence (CsPME1 and CsPME3) and a third lambda clone that contained partial PE sequence (CsPME2) were characterized. The CsPME1 gene contained two exons (1063 and 689 bp) interrupted by a 1452 bp intron, whereas the CsPME3 gene had two exons (844 and 686 bp) interrupted by a 771 bp intron. CsPME1 shared significant sequence similarity with the partial clone CsPME2, including the entire cloned region of the first exon, a large region in the 5′ portion of the intron and the 3′ portion of the second exon, but the 3′ portion of the intron and the 5′ portion of the second exon were dissimilar. Southern blots suggested that Valencia orange has two genes within each PE group. Full-length cDNA clones that shared 99% sequence identity with CsPME1 and CsPME3 were isolated. Both groups of PE genes were differentially expressed in tissues of Valencia orange, and in addition CsPME3 appeared to be ethylene-regulated. The deduced proteins of PE cDNA clones CsPME1 (63.5 kDa) and CsPME3 (56.3 kDa) were considerably larger than the PE protein we isolated from Valencia orange juice vesicles and also other mature plant PE proteins. The estimated size of group I (2.2 kb) and group II (2.0 kb) PE mRNAs also predicted a larger protein than was isolated from juice vesicles. Alignment of the mature tomato and mung bean PE proteins, the most N-terminal sequence we obtained from polypeptides derived from the 36 kDa PE isolated from juice vesicles and the deduced amino acid sequences of plant PE cDNA clones suggest that a post-translational cleavage event separates the variable N-terminus from the more conserved C-terminal domain of the mature PE protein.     

Organization of the mouse microfibril-associated glycoprotein-2 (MAGP-2) gene

A 1.4-kb EST clone encoding mouse microfibril-associated glycoprotein-2 (MAGP-2), identified by its similarity with the reported human cDNA, was used to screen a mouse 129 genomic bacterial artificial chromosome (BAC) library. The mouse gene contains 10 exons spanning 16 kb, located on the distal region of Chromosome (Chr) 6. The exons range in size from 24 to 963 bp, with the ATG located in exon 2. The tenth and largest exon contains 817 bp of 3′ untranslated sequence, including a B2 repetitive element. Northern analysis demonstrates abundant expression of MAGP-2 mRNA in skeletal muscle, lung, and heart. Sequence analysis of additional cDNA clones suggests that the two mRNA forms of MAGP-2 in the mouse arise from alternative polyadenylation site usage. The promoter does not contain an obvious TATA box, and the sequence surrounding the start site does not conform to the consensus for an initiator promoter element. Additionally, the mouse promoter contains 22 copies of a CT dinucleotide repeat sequence located ∼155 bp 5′ to exon 1. Received: 27 August 1999 / Accepted: 2 November 1999     

Human gastric intrinsic factor: characterization of cDNA and genomic clones and localization to human chromosome 11

A human gastric intrinsic factor (IF) cDNA clone was isolated using a rat cDNA clone as a probe. Comparison of the predicted amino acid sequence revealed 80% identity of human IF with rat IF. These cDNA clones were used to isolate and map two overlapping clones encoding the human IF gene. The first exon of the cloned region (exon 2) contains 30 bp of the 5' untranslated region, the signal peptide, and the first 8 amino acids of the mature protein. Exons 3-10 encode the remainder of the coding and 3' noncoding regions. Southern analysis of genomic DNA indicated the presence of a single human IF gene and also revealed the presence of strong hybridizing sequences in genomic DNA from monkey, rat, mouse, cow, and human, suggesting that the IF gene is well conserved. The IF gene was localized to human chromosome 11 by concurrent cytogenetic and cDNA probe analysis of a panel of human X mouse somatic cell hybrids. Southern analysis of genomic DNA from patients with congenital pernicious anemia (lacking intrinsic factor) revealed normal restriction fragment patterns, suggesting that a sizable gene deletion was not responsible for the deficiency.     

Two alternative exons can result from activation of the cryptic splice acceptor site deep within intron 2 of the dystrophin gene in a patient with as yet asymptomatic dystrophinopathy

Intron 2 of the dystrophin gene is unusually large, extending 157 kb on the X-chromosome, and is known to contain one cryptic exon 2a. Here, we report that a single nucleotide change in the middle of this huge intron is a source of two novel extra exons. A novel point mutation changing T to A nucleotide was identified at 5591 bp downstream from the 3' end of exon 2 (T310+5591A) in genomic DNA of an asymptomatic dystrophinopathy case. The mutation identification was initiated by detection of two novel dystrophin mRNAs containing a 132-nucleotide or 46-nucleotide insertion between exons 2 and 3 in lymphocytes but one with a 132-nucleotide insertion in skeletal muscle. It was concluded that T310+5591A created a novel consensus sequence for a splice acceptor site leading to the formation of two novel exon structures by using two cryptic splice donor sites at 132 bp or 46 bp downstream. The former maintained the dystrophin reading frame and was expected to insert 44 amino acids in the N-terminal domain of dystrophin, whereas the latter created a premature stop codon. An immunohistochemical study of the skeletal muscle of the patient disclosed that the N-terminal domain of dystrophin was not stained, but the rod- and C-terminal domains were stained in a patchy and discontinuous manner, indicating that the in-frame mRNA was functional. Creation of a splice acceptor site by a single nucleotide change leading to extra exon structures is a novel molecular mechanism in human disease.     

Organization of the gene for gelatin-binding protein (GBP28)

GBP28 is a novel human plasma gelatin-binding protein that is encoded by apM1 mRNA, expressed specifically in adipose tissue. Three overlapping clones (two lambda clones and one BAC clone) containing the human plasma gelatin-binding protein (GBP28) gene were isolated and characterized. The GBP28 gene spans 16kb and is composed of three exons from 18bp to 4277bp in size with consensus splice sites. The sizes of the two introns were 0.8 and 12kb, respectively. The gene's regulatory sequences contain putative promoter elements, but no typical TATA box.The third exon of this gene contains a long 3'-untranslated sequence containing three Alu repeats. The exon-intron organization of this gene was very similar to that of obese gene, encoding leptin. We also report the chromosome mapping of this gene by fluorescence in situ hybridization (FISH) using a genomic DNA fragment as a probe. The GBP28 gene was located on human chromosome 3q27. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers ABO12163, ABO12164 or ABO12165.     

Primary structure of pregnancy zone protein. Molecular cloning of a full-length PZP cDNA clone by the polymerase chain reaction

A full-length cDNA clone of the human pregnancy zone protein (PZP) was cloned from the hepatocellular carcinoma cell line Hep3B. Based on the exon sequences of the PZP gene (Devriendt et al. (1989) Gene 81, 325-334; Marynen et al., unpublished data), primer pairs were designed to amplify six overlapping fragments of the PZP cDNA. The obtained cDNA is 4609 bp long and contains an open reading frame coding for 1482 amino acids, including a signal peptide of 25 amino acid residues. Comparison with the published partial PZP amino acid sequence (Sottrup-Jensen et al. (1984) Proc. Natl. Acad. Sci. USA 81, 7353-7357) and the PZP genomic sequences confirmed the identity as a PZP cDNA. 71% of the corresponding amino acid residues in PZP and human alpha 2-macroglobulin (alpha 2M) are identical and all cysteine residues are conserved. A typical internal thiol ester site and a bait domain were identified. A Pro/Thr polymorphism was identified at amino acid position 1180, and an A/G nucleotide polymorphism at bp 4097.     

A discordance between the human muscle NCAM sequence and those seen in other NCAM cDNA clones.

A cDNA clone, designated NC7, has been isolated from human foetal kidney that partially codes for the 140 kDa isoform of human NCAM. This clone contains a 6 bp insert that is not present in the human muscle cDNA clone lambda 4.4. This same sequence has also been found in both a cDNA clone obtained from a human Small Cell Lung Carcinoma (SCLC) line and in human genomic DNA. Furthermore, an equivalent sequence to the 6 bp region identified in the above samples is present in mouse, rat and chicken NCAM. The 6 bp insertion does not lie at a predicted intron/exon boundary as extrapolated by homology studies with the chicken and, therefore, the mechanism by which the sequence is deleted from the human muscle clone lambda 4.4 remains unclear.     

Alternatively spliced RNAs encode several isoforms of CD46 (MCP), a regulator of complement activation

Five alternative cDNA clones were isolated for CD46, also known as the membrane cofactor protein (MCP) for the factor I-mediated cleavage of the complement convertases. One of these cDNA clones (a) was identical to an earlier MCP clone. The other four CD46 clones 3ontained the four NH₂-terminanl short consensus repeat (SCR) units of MCP, but differed at the region encoding the carboxyl-terminal of the protein which includes an extracellular segment rich in Ser, Thr, and Pro residues, a hydrophobic membrane-spanning domain, and a 33 amino acid cytoplasmic tail. The different CD46 cDNAs have variously: (b) inserted a 93 base pair (bp) exon resulting in a new cytoplasmic tail of 26 amino acids; (c) deleted a 42 bp exon from the extracellular Ser/Thr rich region; (d) used a cryptic splice acceptor sequence to delete 37 bp from an exon encoding transmembrane sequence; or (e) failed to splice the intron after the four SCR units. These were shown by northern blot and polymerase chain reaction to arise by alternative splicing of CD46 RNA. Forms (a), (b), and (c) of CD46 RNA are common in placental RNA, but (d) was rare, and (e) was incompletely processed and therefore aberrant. The polymerase chain reaction (PCR) was used to map the sites of the intron/exon junctions and demonstrate further possible splice variants of CD46. The alternative RNAs for CD46 may correlate to the different isoforms of CD46 found in different tissues, tumors, and in serum.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M58050. Address correspondence and offprint requests to: D. F. J. Purcell.     

Complete nucleotide sequence of the high molecular weight human IGF-I mRNA

IGF-I gene expression in mammals typically results in multiple mRNA species ranging in size between 0.7 and 7.6 kb. The smaller mRNA species have largely been characterized by the analysis of nearly full-length cDNAs. This report describes the first complete sequence of the prominent high molecular weight (7.6 kb) IGF-I mRNA species. Isolation and nucleotide sequence analysis of cDNA clones from human adult liver and uterus leiomyoma cDNA libraries resulted in a 7236 bp long sequence followed by a poly(A) tail. The sequence data, in combination with structural analysis of the human IGF-I gene, show that the 7.6 kb human IGF-I mRNA contains 6611 bp of untranslated 3' terminal sequence derived from a single exon. Alternate employment of two polyadenylation signals within the sequence transcribed from this exon generates two mRNAs of 1.1 and 7.6 kb.     

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.     

Characterization of the cytochrome P-450IID subfamily in bovine liver. Nucleotide sequences and microheterogeneity.

To elucidate the molecular mechanisms underlying drug detoxification, the structures of the members of the microsomal cytochrome P-450IID subfamily were analyzed by isolating, mapping and sequencing cytochrome P-450IID (CYP2D) cDNA clones from bovine liver. The screening was performed under nonstringent conditions so that most of the P-450IID subfamily members could be obtained. 114 of the 147 positive clones were classified into four groups on the basis of their restriction-enzyme maps. The maps of the four groups were highly similar, however, the clones of one group contained an insertion of approximately 500 bp in the coding region. Analysis of partial nucleotide sequences of several representative clones from each group showed that the bovine P-450IID subfamily in liver consisted of several, not many, highly similar members, differing by less than 7% in their nucleotide sequences. The location of the insertion found in the minor group corresponded to intron 7 and the GT/AG rule was found at the exon/intron boundary, suggesting that intron 7 was retained in this group. The complete nucleotide sequences of two clones from the major group were examined to determine the structures of the P-450IID subfamily in bovine liver. A full-length cDNA clone (1615 bp) and a partial cDNA clone (1538 bp) contained open reading frames encoding 500 and 487 amino acid residues, respectively. The partial clone lacked the nucleotide sequence corresponding to the first 13 N-terminal amino acid residues. The deduced amino acid sequences of the two clones were 98% similar, and 80% and 68% similar to those from human CYP2D6 and rat CYP2D1, respectively. Comparisons of the amino acid sequences of the P-450IID subfamily members showed the highly conserved C-terminal region of their molecules and the high similarity between the members in one species, especially in cattle and man.     

Identification of a putative gamma-aminobutyric acid (GABA) receptor subunit rho2 cDNA and colocalization of the genes encoding rho2 (GABRR2) and rho1 (GABRR1) to human chromosome 6q14-q21 and mouse chromosome 4.

Screening of a genomic DNA library with a portion of the cDNA encoding the gamma-aminobutyric acid (GABA) receptor subunit rho1 identified two distinct clones. DNA sequencing revealed that one clone contained a single exon from the rho1 gene (GABBR1) while the second clone encompassed an exon with 96% identity to the rho1 gene. Screening of a human retina cDNA library with oligonucleotides specific for the exon in the second clone identified a 3-kb cDNA with an open reading frame of 1395 bp. The predicted amino acid sequence of this cDNA demonstrates 30 to 38% similarity to alpha, beta, gamma, and delta GABA receptor subunits and 74% similarity to the GABA rho1 subunit suggesting that the newly isolated cDNA encodes a new member of the rho subunit family, tentatively named GABA rho2. Polymerase chain reaction (PCR) amplification of rho1 and rho2 gene sequences from DNA of three somatic cell hybrid panels maps both genes to human chromosome 6, bands q14 to q21. Tight linkage was also demonstrated between restriction fragment length variants (RFLVs) from each rho gene and the Tsha locus on mouse chromosome 4, which is homologous to the CGA locus on human chromosome 6q12-q21. These two lines of evidence confirm that GABRR1 and newly identified GABRR2 map to the same region on human chromosome 6. This close physical association and high degree of sequence similarity raises the possibility that one rho gene arose from the other by duplication.     

Cloning, genomic sequence, and chromosome mapping of Scyb11, the murine homologue of SCYB11 (alias betaR1/H174/SCYB9B/I-TAC/IP-9/CXCL11)

A T-cell attracting CXC chemokine phylogenetically related to MIG and SCYB10 was recently characterized and termed SCYB11 (alias betaR1/H174/SCYB9B/I-TAC/IP-9/CXCL11). Here, we cloned the cDNA of the murine homologue of this protein, Scyb11, from interferon-gamma/lipopolysaccharide-stimulated RAW264.7 mouse macrophage-like cells. The nucleotide sequence of Scyb11 shares 63% identity with its human counterpart. It encodes a 100 amino acid immature protein of 11,265 Da which contains a putative signal peptide of 21 amino acids. The molecular mass of the mature protein was calculated to be 9,113 Da. Sequence identity of the murine and human SCYB11 proteins is 68%. Phylogenetic tree analysis of mouse CXC chemokines places SCYB11 together with the murine homologues of MIG and SCYB10 (Crg-2/muIP-10) on an individual branch. A genomic sequence was obtained by genome walking and subcloning DNA fragments from a BAC clone containing Scyb11. Like human SCYB11, Scyb11 contains 4 exons with intron/exon boundaries at positions comparable to the human gene. Whereas introns 2 and 3 are of similar length in the murine and human genes, intron 1 of Scyb11 contains 1,260 bp more than intron 1 of the human gene. Intron 1 of Scyb11 is also characterized by a 201-bp stretch with repetitive sequences of high cryptic simplicity. Using a BAC clone containing Scyb11, this gene could be mapped to chromosome 5 at position 5E3. Since human SCYB11 is localized on 4q21.2, this result confirms the mouse/human homology of the two chromosome regions.     

Isolation of a full-length cDNA encoding mouse aromatase P450

A full-length cDNA clone for aromatase P450 has been isolated from a pregnant mouse ovarian cDNA library. The insert of this clone (2394 bp) contains a 1509-bp open reading frame encoding 503 amino acid residues together with a 46-bp 5'-untranslated stretch and an 839-bp 3'-untranslated region to which a poly(A) tract is attached. Northern blot analysis of ovarian RNA from pregnant mice reveals a major mRNA band of 2.5 kb with a minor band of 2.1 kb. Comparison of mouse aromatase P450 with that of rat, human, and chicken shows 91, 81, and 69% identity in the nucleotide sequence and 92, 79, and 69% identity in the deduced amino acid sequence, respectively. The membrane-spanning domain of mouse aromatase P450 is estimated to be an extremely hydrophobic segment located within the N-terminal region of the molecule. Furthermore, a highly conserved heme-binding domain is noticed.