Genomic cloning of the gene for an IgE-binding lectin reveals unusual utilization of 5' untranslated regions.

epsilon BP (for epsilon binding protein) is a M(r) 31,000 S-type animal lectin that binds to IgE and has been identified as the homologue of Mac-2, a macrophage cell-surface marker, as well as the lectins RL-29, CBP35, and L-34. The protein is composed of two domains with the amino-terminal portion containing tandem repeats of nine amino acids and the carboxyl-terminal half containing consensus sequences shared by S-type animal lectins. We determined the genomic map in both rat and mouse and isolated overlapping genomic clones that contain the 5' two-thirds of the murine gene. The remaining portion of the gene was obtained by polymerase chain reaction (PCR) amplification of genomic murine DNA followed by subcloning into plasmid vectors. The epsilon BP gene is composed of six exons separated by five introns. The entire amino-terminal repetitive sequence is contained in exon III, and the carboxyl-terminal domain is encoded by the three succeeding exons (IV, V, VI). The latter three exons correspond well in size and share sequence homology with three exons coding for 14-kDa S-type lectins. The sequence in exon I offers an explanation for the generation of two mRNAs differing only in their 5' untranslated sequences, previously reported in Mac-2 cDNA clones. Using cDNA synthesis and PCR amplification, we determined that two alternative splice sites are used in many different types of cells. This alternative splicing results in different 5' untranslated regions of the murine epsilon BP mRNA.     

The sedlin gene for spondyloepiphyseal dysplasia tarda escapes X-inactivation and contains a non-canonical splice site

Mutations in the sedlin gene cause spondyloepiphyseal dysplasia tarda (SEDT), a rare X-linked chondrodysplasia. Affected males suffer short stature, deformation of the spine and hips, and deterioration of intervertebral discs with characteristic radiographic changes in the vertebrae. We have sequenced two full-length cDNA clones corresponding to the human sedlin gene. The longest cDNA is 2836 bp, containing a 218 bp 5' untranslated region, a 423 bp coding region, and a 2195 bp 3' untranslated region. The second cDNA does not contain exon 2, suggesting alternative splicing. Sedlin was finely mapped in Xp22.2 by Southern blot analysis on a yeast artificial chromosome/bacterial artificial chromosome map. Comparison of the cDNA sequence and genomic sequence identified six sedlin exons of 67, 142, 112, 147, 84, and 2259 bp. The corresponding introns vary in size from 339 to 14,061 bp. Splice site sequences for four of the five introns conform to the GT/AG consensus sequences, however, the splice site between exons 4 and 5 displays a rare non-canonical splice site sequence, AT/AC. Northern blot analysis showed expression of the sedlin gene in all human adult and fetal tissues tested, with the highest levels in kidney, heart, skeletal muscle, liver, and placenta. Four mRNA sizes were detected with the major band being 3 kb and minor bands of 5, 1.6, and 0.9 kb (the smallest product may reflect a sedlin pseudogene). Sedlin is expressed from both the active and the inactive human X chromosomes helping to explain the recessive nature and consistent presentation of the disease. Human sedlin shows homology to a yeast gene, which conditions endoplasmic reticulum/golgi transport. Characterization of the human sedlin cDNA and determination of the sedlin gene structure enable functional studies of sedlin and elucidation of the pathogenesis of SEDT.     

Nucleotide sequence and genomic organization of a human T lymphocyte serine protease gene

We have determined the nucleotide sequence of 4508 base pairs of human genomic DNA which contain the human serine esterase gene from cytotoxic T lymphocytes (SECT) (equivalent to the 1-3E cDNA clone) and include 879 bp of 5' flanking DNA and 393 bp of 3' flanking DNA. The gene consists of five exons of 88, 148, 136, 261, and 257 nucleotides separated by four introns of 1043, 455, 205, and 643 nucleotides. The location of introns with respect to protein coding sequences in the SECT gene is identical to that of the human cathepsin G and murine granzyme B genes. Comparison of SECT gene exonic sequences to murine granzyme B-F cDNA sequences indicates similarities of 75 and 72% for granzymes B and C and 61, 59, and 61% for granzymes D, E, and F, respectively. The 5' flanking sequence of the SECT gene showed similarity only to the 5' flanking sequence of the murine granzyme B gene, indicating that these genes are homologous. Comparison of the SECT gene sequence to the human cathepsin G sequence indicated no similarity in the 5' flanking DNA although the exonic sequences show 64% sequence similarity overall and 45% sequence similarity in the respective 3' untranslated regions. These similarities suggest that the SECT and cathepsin G genes are members of the same family of serine protease genes. Evidence from high and low stringency Southern transfer analysis of human genomic DNA indicates the presence of another gene of at least 85% sequence similarity to the SECT gene.     

The tyrosinase-related protein-1 gene has a structure and promoter sequence very different from tyrosinase.

We have determined the exon structure of the mouse tyrosinase-related protein-1 (TRP-1) gene. The gene is only 15kb in length, but contains seven introns, in contrast to the tyrosinase gene which is almost 100kb long with only four introns. Only two introns are located in homologous positions in both genes. Intron I of TRP-1 has three alternative 5' splice sites clustered within 21bp, which all splice to the same 3' site. Intron V has a very unusual 5' splice site, which has the dinucleotide GC rather than the conventional GT. We show that as little as 370bp of 5'-flanking DNA is sufficient to direct cell-specific expression of the chloramphenicol acetyl transferase gene. The flanking DNA of TRP-1, unlike tyrosinase, does not contain a TATA box or a CCAAT box. Both mouse genes, however, share an 11bp sequence, also found in human tyrosinase, which we suggest may be a melanocyte-specific promoter element.     

Structure and sequence of the human homeobox gene HOX7.

A cosmid containing the human sequence HOX7, homologous to the murine Hox-7 gene, was isolated from a genomic library, and the positions of the coding sequences were determined by hybridization. DNA sequence analysis demonstrated two exons that code for a homeodomain-containing protein of 297 amino acids. The open reading frame is interrupted by a single intron of approximately 1.6 kb, the splice donor and acceptor sites of which conform to known consensus sequences. The human HOX7 coding sequence has a very high degree of identity with the murine Hox-7 cDNA. Within the homeobox, the two sequences share 94% identity at the DNA level, all substitutions being silent. This high level of sequence similarity is not confined to the homeodomain; overall the human and murine HOX7 gene products show 80% identity at the amino acid level. Both the 5' and 3' untranslated regions also show significant similarity to the murine gene, with 79 and 70% sequence identity, respectively. The sequence upstream of the coding sequence of exon 1 contains a GC-rich putative promoter region. There is no TATA box, but a CCAAT and numerous GC boxes are present. The region encompassing the promoter region, exon 1, and the 5' region of exon 2 have a higher than expected frequency of CpG dinucleotides; numerous sites for rare-cutter restriction enzymes are present, a characteristic of HTF islands.     

Nucleotide sequence of a protamine component CII gene of Salmo gairdnerii.

We have isolated, using nick-translated cloned protamine cDNA's as probes, several genomic clones containing protamine gene sequences from a Charon 4A library of Eco R1 digested rainbow trout (Salmo gairdnerii) DNA. One clone was chosen for detailed study and the 2.5 kbp Bam HI-Eco R1 restriction fragment containing the gene was subcloned in the plasmid pBR322. A 920 bp Bg1 II - Bam HI restriction fragment contains a sequence coding for protamine component CII as well as regions 5' and 3' to the mRNA coding portion. Present in the region 5' to the mRNA coding sequence are the promoter associated signals "TATA" box and "CAAT" box. The 5' untranslated region of the mRNA whose length and sequence were not established from the cDNA clones (1) was determined by nuclease mapping and starts within a sequence similar to the "capping signal" found in other genes. The protamine gene for CII contains no introns, a situation common to most histone genes, but, unlike the histone genes does not occur close to other protamine genes in a "cluster".     

Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing

We have isolated and characterized two distinct myosin heavy chain cDNA clones from a neonatal rat aorta cDNA library. These clones encode part of the light meromyosin region and the carboxyl terminus of smooth muscle myosin heavy chain. The two rat aorta cDNA clones were identical in their 5' coding sequence but diverged at the 3' coding and in a portion of the 3' untranslated regions. One cDNA clone, RAMHC21, encoded 43 unique amino acids from the point of divergence of the two cDNAs. The second cDNA clone, RAMHC 15, encoded a shorter carboxyl terminus of nine unique amino acids and was the result of a 39 nucleotide insertion. This extra nucleotide sequence was not present in RAMHC21. The rest of the 3' untranslated sequences were common to both cDNA clones. Genomic cloning and DNA sequence analysis demonstrated that an exon specifying the 39 nucleotides unique to RAMHC15 mRNA was present, together with the 5' upstream common exons in the same contiguous stretch of genomic DNA. The 39 nucleotide exon is flanked on either side by two relatively large introns of approximately 2600 and 2700 bases in size. RNase protection analysis indicated that the two corresponding mRNAs were coexpressed in both vascular and non-vascular smooth muscle tissues. This is the first demonstration of alternative RNA processing in a vertebrate myosin heavy chain gene and provides a novel mechanism for generating myosin heavy chain protein diversity in smooth muscle tissues.     

Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin

We have isolated cDNA clones generated from the mRNA encoding the opsin apoprotein of bovine rhodopsin and used these cDNAs to isolate genomic DNA clones containing the complete opsin gene. Nucleotide sequence analysis of the cloned DNAs has yielded a complete amino acid sequence for bovine rhodopsin and provided an intron-exon map of its gene. The mRNA homologous sequences in the 6.4 kb gene consist of a 96 bp 5' untranslated region, a 1044 bp coding region, and a surprisingly long approximately 1400 bp 3' untranslated region, and are divided into five exons by four introns that interrupt the coding region. Secondary structure analysis predicts that the bovine rhodopsin chain, like that of bacteriorhodopsin, contains seven transmembrane segments. Interestingly, three of the four introns are immediately distal to the codons for three of these segments, and one of these introns marks the boundary between the C-terminal domain and a transmembrane domain.     

Structure of the gene encoding potato cytosolic pyruvate kinase.

The polymerase chain reaction (PCR) has been used to generate a series of overlapping genomic clones representing 43 bp of 5' untranslated sequence, 63 bp of 3' untranslated sequence and the entire coding sequence of the gene encoding potato cytosolic pyruvate kinase (PKc). This portion of the gene is approximately 4.5 kb in length and is interrupted by three introns, one of which is present in the 5' untranslated region. Southern blot analysis indicates that PKc is encoded by a small gene family, and sequence data from a number of PCR-derived genomic clones indicate that there are as many as six PKc genes. Sequence differences between the PCR-generated genomic clones and a PKc cDNA clone are discussed with respect to the fidelity of Taq polymerase. An alignment of intron placement in the potato PKc gene with intron placement in PK genes from other sources indicates that two of the potato introns correspond to intron positions in other species.     

Isolation and characterization of related cDNA clones encoding skeletal muscle beta-tropomyosin and a low-molecular-weight nonmuscle tropomyosin isoform.

We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.     

Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.     

Evolution of the functional human beta-actin gene and its multi-pseudogene family: conservation of noncoding regions and chromosomal dispersion of pseudogenes.

We have assigned six members of the human beta-actin multigene family to specific human chromosomes. The functional gene, ACTB, is located on human chromosome 7, and the other assigned beta-actin-related sequences are dispersed over at least four different chromosomes including one locus assigned to the X chromosome. Using intervening sequence probes, we showed that the functional gene is single copy and that all of the other beta-actin related sequences are recently generated in evolution and are probably processed pseudogenes. The entire nucleotide sequence of the functional gene has been determined and is identical to cDNA clones in the coding and 5' untranslated regions. We have previously reported that the 3' untranslated region is well conserved between humans and rats (Ponte et al., Nucleic Acids Res. 12:1687-1696, 1984). Now we report that four additional noncoding regions are evolutionarily conserved, including segments of the 5' flanking region, 5' untranslated region, and, surprisingly, intervening sequences I and III. These conserved sequences, especially those found in the introns, suggest a role for internal sequences in the regulation of beta-actin gene expression.     

Expression profile and characterisation of a truncated KCNQ5 splice variant

Ion channels encoded by KCNQ genes (1-5) are key regulators of membrane properties in many cell types. The KCNQ5 gene was the last to be identified and has three splice variants that are expressed in human brain and skeletal muscle. The KCNQ5 encoded channel possesses M-current properties and so far no channelopathy has been associated with any of the three variants. We now show that only the shortest KCNQ5 variant, which has exon 9 deleted, was expressed in a variety of murine vascular smooth muscle. In Xenopus oocytes, this variant generated currents with amplitudes, activation kinetics and biophysical properties similar to the full-length variant normally expressed in neuronal tissue. Furthermore sensitivity to block by XE991 and activation by retigabine were also similar between both variants. These data represent an exhaustive characterisation of a truncated KCNQ5 splice variant that may contribute to the native XE991-sensitive channel in murine vasculature.