Partial structure of the gene for chicken cartilage proteoglycan core protein

A genomic DNA fragment (gCORE-1), encoding a portion of the cartilage proteoglycan core protein, has been isolated from a phage library using cDNA as a probe. The genomic insert is about 17 kilobase pairs; two BamHI fragments of the insert (1.3 and 4.8 kilobase pairs) contain most of the hybridizable sequences found in the cDNA. Sequence analysis of these fragments shows that they contain a total of five exons that encompass 216 amino acid residues, all of which are identical to those of the corresponding cDNA sequence. Three of the exons, which are adjacent to one another, are very similar to the corresponding exons in the gene of a rat hepatic lectin as well as to an exon in the gene of human pulmonary surfactant-associated protein. There is a strong degree of conservation of amino acid sequences encoded in the three genes, although there is no similarity between their introns. The sizes of the five exons in gCORE-1, except for one (which is indeterminate because only a partial cDNA sequence is available), are less than 184 base pairs, whereas the sizes of the introns range from 218 to greater than 2629 base pairs. Four of the introns interrupt an exon codon at either their donor or acceptor sites, between the first and second nucleotides. Only one intron does not split a codon. Intron and exon boundary sites are in agreement with known consensus sequences for introns. The dispersed distribution and relatively small size of the exons, if representative of the entire gene, suggest that the complete gene which codes for the core protein may be quite sizable.     

Molecular Characterization of Duplicate Cytosolic Phosphoglucose Isomerase Genes in Clarkia and Comparison to the Single Gene in Arabidopsis

The nucleotide sequence of PgiC1-a which encodes a cytosolic isozyme of phosphoglucose isomerase (PGIC; EC 5.3.1.9) in Clarkia lewisii, a wildflower native to California, is described and compared to the previously published sequence of the duplicate PgiC2-a from the same genome. Both genes have the same structure of 23 exons and 22 introns located in identical positions, and they encode proteins of 569 amino acids. Exon and inferred protein sequences of the two genes are 96.4% and 97.2% identical, respectively. Intron sequences are 88.2% identical. The high nucleotide similarity of the two genes is consistent with previous genetic and biosystematic findings that suggest the duplication arose within Clarkia. A partial sequence of PgiC2-b was also obtained. It is 99.5% identical to PgiC2-a in exons and 99.7% in introns. The nucleotide sequence of the single PgiC from Arabidopsis thaliana was also determined for comparison to the Clarkia genes. The A. thaliana PgiC has 21 introns located at positions identical to those in Clarkia PgiC1 and PgiC2, but lacks the intron that divides Clarkia exons 21 and 22. The A. thaliana PGIC protein is shorter, with 560 amino acids, and differs by about 17% from the Clarkia PGICs. The PgiC in A. thaliana was mapped to a site 20 cM from restriction fragment length polymorphism marker 331 on chromosome 5.     

A canonical sequence organization at the 5'-end of the myosin heavy chain genes

The sequences encoding the 5'-ends of three chicken fast-white myosin heavy chain (MHC) genes have been determined. When compared with the sequences of two other MHC genes it is apparent that both the exon and intron positions are conserved. All exon sequences are highly conserved; there is absolute amino acid conservation in the second and third exons. In addition, while the first and third introns diverge among the genes, the second intron is highly conserved between the five. This intron contains a 24-bp sequence that is repeated twice in one of the introns and once in the other four. Analyses indicate that this sequence, which is partially homologous to 7SL RNA, appears to be largely restricted to the MHC gene family. Analysis of the 5'-flanking sequences show that while small homologies are present between some of the genes, they have extensively diverged in this region.     

Sequence of the chicken delta 2 crystallin gene and its intergenic spacer. Extreme homology with the delta 1 crystallin gene

The chicken delta-crystallin locus consists of 2 nonallelic, tandemly arranged genes (5'-delta 1-delta 2-3'). Only the delta 1 gene is known to be expressed. The nucleotide sequence for the delta 1 gene has been reported recently (Nickerson, J.M., Wawrousek, E.F., Hawkins, J.W., Wakil, A.S., Wistow, G.J., Thomas, G., Norman, B.L., and Piatigorsky, J. (1985) J. Biol. Chem. 260, 9100-9105; Ohno, M., Sakamoto, H., Yasuda, K., Okada, T.S., and Shimura, Y. (1985) Nucleic Acids Res. 13, 1593-1606). We now report the sequence for the delta 2 gene and the 4-kilobase intergenic spacer between the two delta-crystallin genes. The delta 2 gene, like the delta 1 gene, has 17 exons and 16 introns. The homologous exons are remarkably similar: exons 3-17 are identical in size between delta 1 and delta 2, and the sequence homology ranges from 70% (exon 2) to 100% (exons 7, 12, and 15), with the remaining exons having 89-98% identity between the delta 1 and delta 2 genes. Consequently, the encoded delta 2 polypeptide is 91% identical to the delta 1 polypeptide. Considerable similarity also exists between homologous introns of delta 1 and delta 2, with most of the differences accounted for by insertions and/or deletions. The presence of a TATA box, consensus splice junctions (almost identical to the delta 1 gene), lariat branch sequences, and a polyadenylation signal strengthen the possibility that delta 2 is a functional gene.     

Concerted and divergent evolution within the rat gamma-crystallin gene family

The nucleotide sequences of six rat gamma-crystallin genes have been determined. All genes have the same mosaic structure: the first exons contain a relatively short (25 to 44 base-pair) 5' non-coding region and the first nine base-pairs of the coding sequence, the second exons encode protein motifs I and II, while protein motifs III and IV are encoded by the third exons. The third exons also contain a 60 to 67-base-pair long 3' non-coding region. In the gamma 1-2 gene, the splice acceptor site of the third exon has been shifted three base-pairs upstream. Hence, the protein product of this gene is one amino acid residue longer. The first introns, though varying in length from 85 to 100 base-pairs, are conserved in sequence. The second introns vary considerably in length (0.9 X 10(3) to 1.9 X 10(3) base-pairs) and sequence. The second exons of the genes show concerted evolution and have undergone multiple gene conversions. In contrast, the third exons show divergent evolution. From the sequences of the third exons, an evolutionary tree of the gene family was constructed. This tree suggests that three of the present genes derive directly from the genes that originated from a tandem duplication of a two-gene cluster. Two duplications of the last gene of the four-gene cluster then yielded the other three genes. Region a' of the third exon, encoding protein motif III, is variable, while the region encoding protein motif IV (b') is constant. We postulate that this variability in region a' is due to a period of radiation after each gene duplication. A comparison of the rat sequences with those of orthologous sequences from other species shows that the variation in region a' is now preserved. Hence, it might specify the specific functional property of each gamma-crystallin protein within the lens.     

Structural organization and complete sequence of the human alpha-N-acetylgalactosaminidase gene: homology with the alpha-galactosidase A gene provides evidence for evolution from a common ancestral gene.

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc; EC 3.2.1.49), the lysosomal glycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties in glycoconjugates, is encoded by a gene localized to chromosome 22q13----qter. The deficient activity of this enzyme results in Schindler disease, an autosomal recessive disorder characterized by the increased urinary excretion of glycopeptides and oligosaccharides containing alpha-N-acetylgalactosaminyl moieties. Recently, the 3.6-kb full-length alpha-GalNAc cDNA sequence was isolated and found to have remarkable nucleotide and predicted amino acid homology (55.8 and 46.9%, respectively) with the human alpha-galactosidase A (alpha-Gal A) cDNA. To investigate the possible evolutionary relatedness of the two glycosidases, the alpha-GalNAc chromosomal gene was isolated and characterized. Screening of a human genomic DNA cosmid library resulted in the identification of a clone, gAGB-1, with an approximately 35-kb insert that contained the entire alpha-GalNAc gene. A single approximately 15-kb EcoRI fragment of gAGB-1, which contained the complete 3.6-kb cDNA sequence, was digested and the subcloned fragments were sequenced in both orientations. The 13,709-bp alpha-GalNAc gene had nine exons ranging from 95 to 2028 bp and intronic sequences of 304 to 2684 bp. All exon/intron junctions conformed to the GT/AG consensus rule. Analysis of 1.4 kb of 5' flanking sequence revealed three Sp1 and two CAAT-like promoter elements. This region was GC-rich (56%), but no HTF island was identified. The gene contained six Alu-repetitive elements, all in the reverse orientation. Comparison of the structural organization of the alpha-GalNAc and the alpha-Gal A genes revealed that all six alpha-Gal A introns were identically positioned in the homologous alpha-GalNAc exonic sequence. Two additional introns, 1 and 8, were identfied in the alpha-GalNAc gene. The predicted amino acid sequences of alpha-GalNAc exons 2 through 7 and those of corresponding alpha-Gal A exons 1 through 6 were 46.2 to 62.7% identical. In contrast, there was little, if any, similarity between the deduced amino acid sequences of alpha-Gal A exon 7 and alpha-GalNAc exons 8 and 9. The remarkable amino acid identity and the identical exonic interruption by six introns of the alpha-GalNAc and alpha-Gal A genes suggest that this region in both genes is evolutionarily related and arose through duplication and divergence from a common ancestral gene.     

Evolution of collagen IV genes from a 54-base pair exon: A role for introns in gene evolution

The exon structure of the collagen IV gene provides a striking example for collagen evolution and the role of introns in gene evolution. Collagen IV, a major component of basement membranes, differs from the fibrillar collagens in that it contains numerous interruptions in the triple helical Gly-X-Y repeat domain. We have characterized all 47 exons in the mouse alpha 2(IV) collagen gene and find two 36-, two 45-, and one 54-bp exons as well as one 99- and three 108-bp exons encoding the Gly-X-Y repeat sequence. All these exons sizes are also found in the fibrillar collagen genes. Strikingly, of the 24 interruption sequences present in the alpha 2-chain of mouse collagen IV, 11 are encoded at the exon/intron borders of the gene, part of one interruption sequence is encoded by an exon of its own, and the remaining interruptions are encoded within the body of exons. In such "fusion exons" the Gly-X-Y encoding domain is also derived from 36-, 45-, or 54-bp sequence elements. These data support the idea that collagen IV genes evolved from a primordial 54-bp coding unit. We furthermore interpret these data to suggest that the interruption sequences in collagen IV may have evolved from introns, presumably by inactivation of splice site signals, following which intronic sequences could have been recruited into exons. We speculated that this mechanism could provide a role for introns in gene evolution in general.     

Cloning of the full-length coding sequence of rat liver-specific organic anion transporter-1 (rlst-1) and a splice variant and partial characterization of the rat lst-1 gene

The full-length coding sequence of rat liver-specific organic anion transporter-1 (lst-1) and its splice variant have been cloned. The full-length rat lst-1 (designated rlst-1a) encodes a protein containing 687 amino acids and has 12-putative transmembrane domains, multiple potential N-glycosylation and phosphorylation sites. Therefore, rat lst-1a has 35 additional amino acid residues compared to the previously reported rat lst-1. A splice variant (designated rlst-1c) reported in this communication encodes a protein containing 654 amino acids and has 10-putative transmembrane domains. PCR analysis suggests that rlst-1a is the most abundant form in liver. Phylogenetic analysis reveals that rat lst-1a is an ortholog of human LST-1 (hLST-1) and mouse lst-1 (mlst-1). The rlst-1 gene is composed of 15 exons and 14 introns. Analysis of exon-intron boundary reveals that the splice variant rlst-1c lacks the entire exon 7, while the previously reported rat lst-1 (designated herein as rlst-1b) lacks approximately half of exon 10, and the splicing has occurred through alternative usage of a splice donor site within exon 10.     

Structure and expression of the gene coding for the human serpin hLS2

We have analyzed genomic clones encoding human leuserpin 2 (hLS2). The gene covers about 14.5 kilobases and consists of 5 exons and 4 introns. The genes coding for hLS2, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and rat angiotensinogen share an equivalent exon-intron structure and therefore constitute a distinct subgroup within the serpin gene family, which otherwise displays a highly variable exon-intron pattern. With the exception of a segment in the second exon, the sequence similarity of the genes coding for hLS2 and alpha 1-antitrypsin extends to all exons including one encoding the 5'-untranslated sequences. The implications of these findings with respect to the genesis of the amino-terminal heterogeneity in the serpin family are discussed.     

Rearrangements of the red-cell membrane glycophorin C (sialoglycoprotein beta) gene. A further study of alterations in the glycophorin C gene.

We have cloned portions of the glycophorin C (sialoglycoprotein beta) gene from individuals with red cells of normal, Gerbich and Yus phenotypes. The clones contain up to three exons of the glycophorin C gene (designated exons 2, 3 and 4). Analysis by restriction mapping and DNA sequencing confirmed that the deletions causing the Gerbich and Yus phenotypes are located entirely within the glycophorin C gene. Sequencing of the normal gene showed that not only do exon 2 and exon 3 have related DNA sequences, but also that both the 5' and 3' flanking intronic DNA sequences are almost identical. The two variant genes each lack a different exon: the Yus type gene lacks exon 2, whereas the Gerbich-type gene lacks exon 3. We suggest that the observed deletions are due to recombination between the regions of homologous intronic repeats. We also provide evidence that an unequal cross-over mechanism may be responsible for a number of observed glycophorin C gene rearrangements, including an insertion mutation in Lewis II (Lsa)-type red cells that has not previously been reported.     

Comparative analysis of the structural organization of two closely related vitellogenin genes in X. laevis

The structural organization of the two closely related vitellogenin genes A1 and A2 has been determined and compared by electron microscopy. In both genes the mRNA-coding sequence of 6 kb is interrupted 33 times, leading to a total gene length of 21 kb for gene A1 and 16 kb for gene A2. Thus both genes have a mean exon length of 0.175 kb, while the mean intron length is 0.45 kb in gene A1 and 0.31 kb in gene A2. Because the introns interrupt the structural sequence at homologous positions in genes A1 and A2, we suggest that these two genes are the products of a duplication of an ancestral gene which had an intron-exon arrangement similar to that of the extant genes. Since the duplication event, the sequence and length of the analogous introns have changed rapidly, whereas homologous exons have diverged to an extent of only 5% of their sequences. The results suggest different mechanisms of evolution for exons and introns. While the exons evolved primarily by point mutations, such mutations, as well as deletion, insertion and duplication events, were important in the evolution of the introns.     

Nucleotide sequence analysis of the human salivary protein genes HIS1 and HIS2, and evolution of the STATH/HIS gene family

Human histatins are a family of low-M(r), neutral to very basic, histidine-rich salivary polypeptides. They probably function as part of the nonimmune host defense system in the oral cavity. A 39-kb region of DNA containing the HIS1 and HIS2 genes was isolated from two human genomic phage libraries as a series of overlapping clones. The nucleotide sequences of the HIS1 gene and part of the HIS2(1) gene were determined. The transcribed region of HIS1 spans 8.5 kb and contains six exons and five introns. The HIS1 and HIS2(1) genes exhibit 89% overall sequence identity, with exon sequences exhibiting 95% identity. The two loci probably arose by a gene duplication event approximately 15-30 Mya. The HIS1 sequence data were also compared with that of STATH. Human statherin is a low-M(r) acidic phosphoprotein that acts as an inhibitor of precipitation of calcium phosphate salts in the oral cavity. The HIS1 and STATH genes show nearly identical overall gene structures. The HIS1 and STATH loci exhibit 77%-81% sequence identity in intron DNA and 80%-88% sequence identity in noncoding exons but only 38%-43% sequence identity in the protein-coding regions of exons 4 and 5. These unusual data suggest that HIS1, HIS2, and STATH belong to a single gene family exhibiting accelerated evolution between the HIS and STATH coding sequences.      

Structure of the chromosomal gene for human serum prealbumin

The human prealbumin gene has been cloned and its complete nucleotide sequence determined. The gene has a size of about 6.9 kb and is composed of four exons and three introns. Two Alu family sequences having opposing polarity were found in introns. In the 5'-flanking region, we found two overlapping sequences which have extensive homology to the glucocorticoid-responsive element. Three sequences identical with the enhancer core sequence were identified in introns and the 3'-flanking region. Unusual tandem repeats of a sequence, TTTTG, were also found in the 5'-flanking region and introns.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. The cytochrome b gene has an intron closely related to the first two introns in the Saccharomyces cerevisiae cox1 gene

The DNA sequence of the cob region of the Schizosaccharomyces pombe mitochondrial DNA has been determined. The cytochrome b structural gene is interrupted by an intron of 2526 base-pairs, which has an open reading frame of 2421 base-pairs in phase with the upstream exon. The position of the intron differs from those found in the cob genes of Saccharomyces cerevisiae, Aspergillus nidulans or Neurospora crassa. The Sch. pombe cob intron has the potential of assuming an RNA secondary structure almost identical to that proposed for the first two cox1 introns (group II) in S. cerevisiae and the p1-cox1 intron in Podospora anserina. It has most of the consensus nucleotides in the central core structure described for this group of introns and its comparison with other group II introns allows the identification of an additional conserved nucleotide stretch. A comparison of the predicted protein sequences of group II intronic coding regions reveals three highly conserved blocks showing pairwise amino acid identities of 34 to 53%. These regions comprise over 50% of the coding length of the intron but do not include the 5' region, which has strong secondary structural features. In addition to the potential intron folding, long helical structures involving repetitive sequences can be formed in the flanking cob exon regions. A comparison of the Sch. pombe cytochrome b sequence with those available from other organisms indicates that Sch. pombe is evolutionarily distant from both budding yeasts and filamentous fungi. As was seen for the Sch. pombe cox1 gene (Lang, 1984), the cob exons are translated using the universal genetic code and this distinguishes Sch. pombe mitochondria from all other fungal and animal mitochondrial systems.     

Characterization of the gene for prostate-specific antigen, a human glandular kallikrein

The gene for the human glandular kallikrein, prostate-specific antigen, has been cloned. The sequence of 7130 nucleotides encompassing the gene and 633 bp of 5' and 639 bp of 3' flanking DNA has been determined. The translation initiation site was slightly heterogeneous, yielding 5' non-translated leader sequences of 41 and 35 bp. The gene is divided into five exons, with introns located at positions identical with those found in other glandular kallikrein genes. The nucleotide sequence is very similar to that of the human kallikrein gene hGK-1, with 76 to 93% of the nucleotides being identical in the exons and 76 to 87% in the introns. The similarity also extends approximately 200 bp into the sequence flanking the 5' end of hGK-1 and several other, both human and rodent, glandular kallikrein genes.     

The nucleotide sequence of bovine MHC class IIDQB andDRB genes

The nucleotide sequences of most of the exons and parts of the introns of twoBoLA-DQB genes and twoBoLA-DRB genes have been determined. The structure of these genes is very similar to that of human major histocompatibility complex (MHC) class II genes. The twoDQB genes probably represent true alleles. Based on the exons sequenced, bothDQB genes and one of theDRB genes seem to be functional. The otherDRB gene is a pseudogene; stopcodons are found in the exons encoding the second and transmembrane domain and, furthermore, a 2 base pair (bp) deletion has occured in the leader exon which places the initiation start codon out of frame. Also in this pseudogene, an almost perfect inverted repeat of 200 bp is found flanking the exon encoding the first domain, which might have been the result of a duplication/inversion event. The sequences presented in this paper do not contain any repetitions. Therefore, DNA fragments containing these sequences can be used as homologous bovine probes in restriction fragment length polymorphism (RFLP) analysis to study disease association in cattle.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M30002–M30014. Address correspondence and offprint requests to: M. A. M. Groenen.     

Analysis of the genomic organization of the human cationic amino acid transporters CAT-1, CAT-2 and CAT-4

Summary. By screening nucleotide databases, sequences containing the complete genes of the human cationic amino acid transporters (hCATs) 1, 2 and 4 were identified. Analysis of the genomic organization revealed that hCAT-2 consists of 12 translated exons and most likely of 2 untranslated exons. The splice variants hCAT-2A and hCAT-2B use exon 7 and 6, respectively. The hCAT-2 gene structure is closely related to the structure of hCAT-1, suggesting that they belong to a common gene family. hCAT-4 consists of only 4 translated exons and 3 short introns. Exons of identical size and highly homologous to exon 3 of hCAT-4 are present in hCAT-1 and hCAT-2. Received September 8, 2000 Accepted January 8, 2001