Structural and evolutionary relationships among five members of the human gamma-crystallin gene family.

We have characterized five human gamma-crystallin genes isolated from a genomic phage library. DNA sequencing of four of the genes revealed that two of them predict polypeptides of 174 residues showing 71% homology in their amino acid sequence; the other two correspond to closely related pseudogenes which contain the same in-frame termination codon at identical positions in the coding sequence. Two of the genes and one of the pseudogenes are oriented in a head-to-tail fashion clustered within 22.5 kilobases. All three contain a TATA box 60 to 80 base pairs upstream of the initiation codon and a highly conserved segment of 44 base pairs in length immediately preceding the TATA box. The two genes and the two pseudogenes are similar in structure: each contains a small 5' exon encoding three amino acids followed by two larger exons that correspond exactly to the two similar structural domains of the polypeptide. The first intron varies from 100 to 110 base pairs, and the second intron ranges from 1 to several kilobases, rendering an overall gene size of 1.7 to 4.5 kilobases. At least one of the two pseudogenes appears to have been functional before inactivation, suggesting that their identical mutation was generated by gene conversion.     

Structure of the murine complement factor H gene

Factor H is a regulatory protein of the alternative pathway of complement activation comprised of 20 tandem repeating units of 60 amino acids each. A factor H cDNA clone was used to identify 17 genomic clones from a cosmid library. Four clones were selected for analysis of intron/exon junctions and 5' and 3' regions of the gene and for mapping of the exons. The factor H gene was found to be comprised of 22 exons. Each repeating unit is encoded by one exon, except the second repeat, which is coded by two exons; the leader sequence is encoded by a separate exon. The exons range in size from 77 to 210 base pairs (bp) and average 178 bp. They span a region of approximately 100 kilobases (kb) on chromosome 1. The leader sequence exon is 26 kb upstream of the first repeat exon, representing the largest intron. The other introns range in size from 86 bp to 12.9 kb, and the average intron size is 4.7 kb. Analysis of the genomic organization of the factor H gene has provided insight into the protein structure and will enable the construction of deletion mutants for functional studies.     

Nucleotide sequence and organization of the human S-protein gene: repeating peptide motifs in the "pexin" family and a model for their evolution

The S-protein/vitronectin gene was isolated from a human genomic DNA library, and its sequence of about 5.3 kilobases including the adjacent 5' and 3' flanking regions was established. Alignment of the genomic DNA nucleotide sequence and the cDNA sequence indicated that the gene consisted of eight exons and seven introns. The intron positions in the S-protein gene and their phase type were compared to those in the hemopexin gene which shares amino acid sequence homologies with transin and the S-protein. Three introns have been found at equivalent positions; two other introns are very close to these positions and are interpreted as cases of intron sliding. Introns 3-7 occur at a conserved glycine residue within repeating peptide segments, whereas introns 1 and 2 are at the boundaries of the Somatomedin B domain of S-protein. The analysis of the exon structure in relation to repeating peptide motifs within the S-protein strongly suggests that it contains only seven repeats, one less than the hemopexin molecule. A very similar repeat pattern like that in hemopexin is shown to be present also in two other related proteins, transin and interstitial collagenase. An evolutionary model for the generation of the repeat pattern in the S-protein and the other members of this novel "pexin" gene family is proposed, and the sequence modifications for some of the repeats during divergent evolution are discussed in relation to known unique functional properties of hemopexin and S-protein.     

A hyaluronan binding link protein gene family whose members are physically linked adjacent to chondroitin sulfate proteoglycan core protein genes: the missing links

We describe a vertebrate hyaluronan and proteoglycan binding link protein gene family (HAPLN), consisting of four members including cartilage link protein. The encoded proteins share 45-52% overall amino acid identity. In contrast to the average sequence identity between family members, the sequence conservation between vertebrate species was very high. Human and mouse link proteins share 81-96% amino acid sequence identity. Two of the four link protein genes (HAPLN2 and HAPLN4) were restricted in expression to the brain/central nervous system, while one of the four genes (HAPLN3) was widely expressed. Genomic structures revealed that all four HAPLN genes were similar in exon-intron organization and were also similar in genomic organization to the 5' exons for the CSPG core protein genes. Strikingly, all four HAPLN genes were located immediately adjacent to the four CSPG core protein genes creating four pairs of CSPG-HAPLN genes within the mammalian genome. Furthermore, the two brain-specific HAPLN genes (HAPLN2 and HAPLN4) were physically linked to the brain-specific CSPG genes encoding brevican and neurocan, respectively. The tight physical association of the HAPLN and CSPG genes supports a hypothesis that the first HAPLN gene arose as a partial gene duplication event from an ancestral CSPG gene. There is some degree of coordinated expression of each gene pair. Collectively, the four HAPLN genes are expressed by most tissue types, reflecting the fundamental importance of the hyaluronan-dependent extracellular matrix to tissue architecture and function in vertebrate species. Comparison of the genomic structures for the HAPLN, CSPG genes and other members of the link module superfamily provide strong support for a common evolutionary origin from an ancestral gene containing one link module encoding exon.     

The main fatty acid-binding protein in the liver of the shark (Halaetunus bivius) belongs to the liver basic type. Isolation, amino acid sequence determination and characterization.

Three fatty acid-binding proteins (FABPs) from the liver of the shark Halaetunus bivius were isolated and characterized: one of them belongs to the liver-type FABP family and the other two to the heart-type FABP family. The complete primary structure of the first FABP, and partial primary structures of the two others, were determined. The liver-type FABP constitutes 69% of the total FABPs, and its amino acid sequence presents the highest identity with chicken, catfish, iguana and elephant fish liver basic FABPs. The L-FABP protein has low affinity for palmitic and oleic acids and high affinity for linoleic and arachidonic acids and other hydrophobic ligands, all of them important for the metabolic functions of the liver. In contrast, both heart-type FABPs have the highest affinity for palmitic acid, the principal fatty acid mobilized from fat deposits for beta-oxidation.     

Molecular evolution of streptococcal M protein: cloning and nucleotide sequence of the type 24 M protein gene and relation to other genes of Streptococcus pyogenes.

The structural gene for the type 24 M protein of group A streptococci has been cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene and the 3' and 5' flanking regions was determined. The sequence includes an open reading frame of 1,617 base pairs encoding a pre-M24 protein of 539 amino acids and a predicted Mr of 58,738. The structural gene contains two distinct tandemly reiterated elements. The first repeated element consists of 5.3 units, and the second contains 2.7 units. Each element shows little variation of the basic 35-amino-acid unit. Comparison of the sequence of the M24 protein with the sequence of the M6 protein (S. K. Hollingshead, V. A. Fischetti, and J. R. Scott, J. Biol. Chem. 261:1677-1686, 1986) indicates that these molecules have are conserved except in the regions coding for the antigenic (type specific) determinant and they have three regions of homology within the structural genes: 38 of 42 amino acids within the amino terminal signal sequence, the second repeated element of the M24 protein is found in the M6 molecule at the same position in the protein, and the carboxy terminal 164 amino acids, including a membrane anchor sequence, are conserved in both proteins. In addition, the sequences flanking the two genes are strongly conserved.     

Structure and chromosome assignment of the murine p36 (calpactin I heavy chain) gene

p36 is a major substrate of both viral and growth factor receptor associated protein kinases. This protein has recently been named calpactin I heavy chain since it is the large subunit of a Ca2(+)-dependent phospholipid and actin binding heterotetramer. The primary structure of p36 has been determined from analysis of cloned cDNA. The protein contains 338 amino acids, has an approximate molecular weight of 39,000, and is comprised of several distinct domains, including four 75 amino acid repeats. From two overlapping cosmid clones isolated from different mouse genomic liver libraries, the complete intron/exon structure of the p36 gene was determined and the 5' and 3' noncoding regions of the gene were analyzed. The coding and 3' untranslated region of the p36 gene contains 12 exons which range in size from 48 to 322 base pairs (bp) with an average size of 107 bp. The repeat structures found at the protein level are not delineated by single exons, but the N-terminal p11-binding domain is encoded by a single exon. Structural mapping of the gene demonstrated that the lengths of the first two introns in the coding region are together approximately 6 kilobases (kb), while the other introns range in size from 600 to 3600 bp with an average size of 1650 bp. The p36 gene is at least 22 kb in length and has a coding sequence of approximately 1 kb, representing only 4.5% of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the human platelet glycoprotein IIIa gene. Comparison with the fibronectin receptor beta-subunit gene

This study was designed to determine the structure of the gene for glycoprotein (GP) GPIIIa, the beta-subunit of the platelet membrane GPIIb-IIIa complex. The complexity of the gene was determined after Southern analysis of human chromosomal DNA. Overlapping genomic clones were isolated from cosmid and phage lambda libraries that contained the entire coding unit of the human gene for the mature GPIIIa protein. The genomic clones spanned approximately 60 kilobase pairs of human DNA sequence. The exon containing segments of the clones was mapped and the exons, including the exonintron junctions, were sequenced. The GPIIIa protein is divided into 14 exons ranging in size from 87 to 430 nucleotides separated by introns, which were 0.3 to 9 kilobase pairs in size. The 3' exon was larger than 1700 nucleotides and contained the 3'-untranslated region. Several structural domains of the GPIIIa protein were contained within individual exons. These included (i) the transmembrane spanning segment, (ii) the cytoplasmic region containing the potential phosphorylation sites, and (iii) the six domains in the NH2-terminal half of GPIIIa that are highly conserved between two other integrin beta-subunits. In contrast, other domains such as the four cysteine-rich repeats were interrupted by introns. Genomic clones for the beta-subunit of the fibronectin receptor (beta 1) were also isolated, partially mapped, and sequenced. Of the eight splice sites identified in beta 1, six occurred at the same amino acid residue in GPIIIa. These results provide genetic evidence that GPIIIa and beta 1 have a common evolutionary origin within the integrin family.     

Heterochromatin protein 1, a known suppressor of position-effect variegation, is highly conserved in Drosophila.

The Su(var)205 gene of Drosophila melanogaster encodes heterochromatin protein 1 (HP1), a protein located preferentially within beta-heterochromatin. Mutation of this gene has been associated with dominant suppression of position-effect variegation. We have cloned and sequenced the gene encoding HP1 from Drosophila virilis, a distantly related species. Comparison of the predicted amino acid sequence with Drosophila melanogaster HP1 shows two regions of strong homology, one near the N-terminus (57/61 amino acids identical) and the other near the C-terminus (62/68 amino acids identical) of the protein. Little homology is seen in the 5' and 3' untranslated portions of the gene, as well as in the intronic sequences, although intron/exon boundaries are generally conserved. A comparison of the deduced amino acid sequences of HP1-like proteins from other species shows that the cores of the N-terminal and C-terminal domains have been conserved from insects to mammals. The high degree of conservation suggests that these N- and C-terminal domains could interact with other macromolecules in the formation of the condensed structure of heterochromatin.     

Isolation of the osteonectin gene: evidence that a variable region of the osteonectin molecule is encoded within one exon

A complementary DNA clone for bovine osteonectin was used to isolate the osteonectin gene from two libraries of bovine genomic DNA fragments. Two overlapping clones were obtained whose relationship was determined by restriction mapping and sequence analysis. The two clones contain the entire osteonectin coding region spanning approximately 11 kilobases of genomic DNA. The coding region of the gene was determined, by electron microscopy and DNA sequencing, to reside in nine exons. In addition, there is at least one 5' exon interrupted by an intron in the 5'-nontranslated sequence of the gene. Excluding this 5' exon and the 3'-terminal exon, the exons are small and approximately uniform in size, averaging 130 +/- 17 base pairs. Three of the exons at the 5' end of the gene were sequenced and appear to encode discrete protein domains. For example, the putative exon 2 contains the coding region for the leader peptide of the molecule. The amino-terminal protein sequence was determined for osteonectin extracted from human, rabbit, and chicken bone and compared with those for bovine, mouse, and pig osteonectin. These data suggest that osteonectin is highly conserved between species, interspecies changes being seen primarily at the amino terminus of the protein and specifically in the region encoded by putative exon 3 in the bovine gene.