Structural organization of multiple rat calmodulin genes

Elsewhere, we have reported the structure of a rat calmodulin gene and two distinct rat calmodulin cDNAs, pRCM1 and pRCM3. Here, I report the cloning and sequencing of the third calmodulin cDNA (pRCM4) and two additional rat calmodulin genes. The original calmodulin gene is named CaM I (pRCM1) and the newly discovered calmodulin genes are named CaM II (pRCM3) and CaM III (pRCM4). CaM II spans about 10 x 10(3) base-pairs and consisted of five exons, while CaM III spans about 7.2 x 10(3) base-pairs and consisted of six exons. One of the introns (intron 3) observed in CaM I and CaM III is lost in CaM II. Otherwise, the intron/exon organization of these genes is exactly the same. In all calmodulin genes, the first intron separates the initiation codon (ATG) from the coding region of the protein. Northern blotting showed that CaM I is transcribed primarily into 1.7 x 10(3) base-pair mRNA in various tissues examined and 4.0 x 10(3) base-pair mRNA mainly in skeletal muscle, CaM II is transcribed into 1.4 x 10(3) base-pair mRNA almost exclusively in brain and CaM III is transcribed predominantly into 2.3 x 10(3) base-pair mRNA and faintly into 1.0 x 10(3) base-pair mRNA mainly in skeletal muscle and brain. DNA sequences in the promoter-regulator regions of these genes are partly homologous but essentially distinct and possess a number of direct repeats, palindromes and feasible stem-loop structures. Together with these, I report here the structures of the third and fourth calmodulin retropseudogenes.     

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.     

Structure and evolution of the heavy chain from rat immunoglobulin E.

The nucleotide sequence of the rat epsilon-chain mRNA has been determined by sequencing cloned cDNA copies of the mRNA. The established sequence covers the coding region, the 3'-non coding region and most of the 5' non-coding region. A comparison with the nucleotide sequence of the human epsilon-chain constant region reveals that C3 and C4 are the most highly conserved domains. The rat epsilon-chain contains a C-terminal decapeptide which is not present in the human counterpart.     

Nucleotide sequence of terminal repeats of 412 transposable elements of Drosophila melanogaster: A similarity to proviral long terminal repeats and its implications for the mechanism of transposition

We have sequenced the long terminal direct repeats (and adjacent DNA) of two members of the 412 family of transposable elements of Drosophila melanogaster cloned on fragments of DNA from strain Oregon R. The repeats of the first element are identical and 481 base-pairs long; the repeats of the second are also identical but are 571 base-pairs long. The first 482 base-pairs of the 571 base-pair sequence correspond to the 481 base-pair repeat differing by five base substitutions and one addition/deletion. The 571 base-pair repeats are rare. Each of these 412 elements is flanked by a four base-pair direct repeat, suggesting that insertion of a 412 element is associated with duplication of four base-pairs. Analysis of the “empty site” from strain Canton S corresponding to one of these elements supports this conclusion. The sequence of 481 base-pair repeats and of 412 DNA immediately adjacent to them show striking similarities to corresponding regions of vertebrate proviruses and we discuss the implications this may have for the mechanism of transposition.     

Structure of rat calmodulin processed genes with implications for a mRNA-mediated process of insertion

Two distinct processed calmodulin genes of rat (lambda SC8 and lambda SC9) were identified, cloned and their DNA sequences determined. The existence of direct repeats of 19 base-pairs for lambda SC8 or 9 base-pairs for lambda SC9 at both ends of the coding plus non-coding regions suggested a possible involvement of a mRNA-mediated process of insertion. Total genomic Southern hybridization suggested the existence of at least three different calmodulin-related genes in the rat genome. The other gene was the bona fide calmodulin gene (lambda SC4) which was split into at least five exons. lambda SC9 contained insertions of one nucleotide and two 17 base-pair direct repeats in the coding region. These insertions cause frameshift mutations probably preventing it from encoding a functional calmodulin. It also carried an insertion of a rat middle repetitive sequence, identifier sequence (IDS: Sutcliffe et al., 1982) in the 3'-non-coding region. Otherwise, it consisted of an almost identical DNA sequence to that of the bona fide calmodulin gene (lambda SC4), including the 3'-non-coding region down to the poly(A) recognition signal, A-A-T-A-A-A. On the other hand, lambda SC8 did not possess frameshift mutations in the coding region, and hence was capable of encoding a functional protein. In fact, a probe specific to the lambda SC8 sequence identified a band in Northern blotting whose size was 300 nucleotides smaller than that of authentic calmodulin mRNA. Comparison of the nucleotide sequences showed that only the coding regions of these two processed genes were homologous, indicating that the divergence of these two processed genes from the common ancestor calmodulin was an ancient event.     

Transposable genetic element found in the 5'-flanking region of the fibroin H-chain gene in a genomic clone from the silkworm Bombyx mori

A transposable genetic element was found in the 5'-flanking region of the fibroin H-chain gene in one of the genomic clones from the silkworm Bombyx mori. This element, named K-1.4, is about 1 X 4 X 10(3) base-pairs long, contains an open reading frame of only 225 base-pairs and has inverted repeats of 12 base-pairs at both ends. Duplication of three base-pairs seems to have occurred when this element was integrated into the silkworm genome. About 15 copies of K-1.4 are present per haploid genome of various silkworm strains. Genomic loci of some of these elements are different among different strains or even among individual offspring of the same parents. K-1.4 is present also in the genome of Bombyx mandarina. The K-1.4-related sequences are present in some species belonging to the family Saturniidae.     

Characterization of a member of the highly repeated long interspersed rat DNA family with long open reading frames

A highly repetitive long interspersed sequence from rat DNA has been isolated and partly characterized. This sequence comprises at least a 1300 base-pair and a 2400 base-pair EcoRI fragment and probably additional elements. The 2400 base-pair segment has been analyzed in detail. It appears to be part of the chromosomal DNA in rat cells. The 2400 base-pair repeat is likely to be distributed over several regions in the rat genome. The 2400 base-pair segment has been cloned, mapped for restriction sites, and part of its nucleotide sequence has been determined. The 2400 base-pair sequence is a member of a typical highly repetitive long interspersed sequence with high copy number and restriction site polymorphism. There are sequence homologies to mouse and human DNA. A striking homology has been detected to the flanking sequences of a repetitive mouse DNA sequence that has been described to be located adjacent to one of the kappa-immunoglobulin variable genes. Elements in the 2400 base-pair rat repeat are transcribed in cells from most rat organs and from several continuous rat cell lines. This RNA from rat cell lines was found polyadenylated or not polyadenylated. The nucleotide sequence of parts of the 2400 base-pair DNA segment revealed open reading frames for polypeptide sequences. Such open reading frames have been detected in two different segments of the 2400 base-pair DNA repeat. Open reading frames exist in the two complementary strands in the same DNA segment. The hypothetical polypeptide whose sequence has been determined in toto has a length of 190 amino acid residues and is enriched in hydrophobic amino acids, reminiscent of the amino acid composition in membrane proteins. Hence, it is conceivable that the 2400 base-pair repeat sequence from rat DNA, at least in part, encodes messenger RNAs that might be translated into functional proteins.     

Conserved structural features are found upstream from the three co-ordinately regulated discoidin I genes of Dictyostelium discoideum

The discoidin I genes of Dictyostelium form a small, co-ordinately regulated multigene family. We have sequenced and compared the upstream regions of the DiscI-alpha, -beta and -gamma genes. For the most part the upstream regions of the three genes are non-homologous. The upstream sequences of the beta and gamma genes are exceedingly A + T-rich, while those of the alpha gene are less so. All three genes have a relatively G + C-rich region 20 to 40 base-pairs in length, found approximately 200 base-pairs 5' to the messenger RNA start site. This G + C-rich region 5' to the beta and gamma genes is flanked by short inverted repeats. Within this region, there is an 11 base-pair exact homology between the alpha and gamma genes, and a less perfect homology between these genes and the beta gene. The homology is flanked at a short distance by interspersed G and T residues. The gamma gene is greater than 90% A + T for greater than 800 base-pairs upstream. Further upstream there is a G + C-rich region that is also found inverted approximately 3.5 X 10(3) base-pairs away. The gamma and beta genes are tandemly linked, and the entire approximately 500 base-pair intergene region between the 3' end of the gamma gene and the 5' end of the beta gene is A + T-rich (approximately 90%) with the exception of the homology region 5' to the gamma gene. We demonstrate also the presence of a discoidin I pseudogene fragment having only 139 base-pairs of discoidin homology with greater than 8% mismatch. It is flanked upstream by five 39 base-pair G + C-rich repeats, and downstream by sequences that are extremely A + T-rich. We discuss the possible significance of the conserved G + C-rich structures on discoidin I gene expression.     

The 1723 element: a long, homogeneous, highly repeated DNA unit interspersed in the genome of Xenopus laevis

We describe a highly repeated DNA element in the Xenopus laevis genome. This sequence, named the 1723 element, was first identified among sequences that are transcribed during embryonic development. The element is present in about 8500 copies per haploid genome, which together accounts for about 2.4% of the genome. Most copies of the element have highly conserved restriction maps, and are interspersed in the genome. The copies range in size from 6000 to 10,000 base-pairs due to an expandable region that contains variable numbers of a tandemly repeating 183 to 204 base-pair unit. The element is framed by an imperfect 18 base-pair inverted sequence, and inverted repeats of 180 to 185 base-pairs are nearby. Sequence analysis of DNA adjacent to three cloned elements shows that the elements are flanked by 8 base-pair direct repeats. These and other properties of 1723 suggest that it may be transposable.     

Complete structure of the hamster alpha A crystallin gene. Reflection of an evolutionary history by means of exon shuffling

The eye lens contains a structural protein, alpha crystallin, composed of two homologous primary gene products alpha A2 and alpha B2. In certain rodents, still another alpha crystallin polypeptide, alpha AIns, occurs, which is identical to alpha A2 except that it contains an insertion peptide between residues 63 and 64. In this paper we describe the complete alpha A crystallin gene that has been cloned from DNA isolated from Syrian golden hamster. Evidence is provided that the alpha A gene is present as a single copy in the hamster genome. The detailed organization of the gene has been established by means of DNA sequence analysis and S1 nuclease mapping, revealing that the gene consists of four exons. The first exon contains the information for the 68 base-pair long 5' non-coding region as well as the coding information for the first 63 amino acids. The second exon encodes the 23 amino acid insertion sequence, the third exon codes for amino acid 87 to 127 of the alpha AIns chain, whereas the last exon encodes the C-terminal 69 amino acids and contains the information for the 523 base-pair long 3' non-coding region. The second exon is bordered by a 3' splice junction (A X G/G X C), which deviates from the consensus for donor splice sites (A X G/G X T). This deviation is found in both hamster and mouse. An internal duplication was detected in the first exon by using a DIAGON-generated matrix for comparison. By means of similar DIAGON-generated matrices it was confirmed that the amino acids coded for by the third and fourth exons are homologous to the small heat-shock proteins of Drosophila, Caenorhabditis and soyabean. The implications of the differential splicing and the evolutionary aspects of the detected homologies are discussed.     

A rapidly evolving region in the immunoglobulin heavy chain loci of rat and mouse: postulated role of (dC-dA)n.(dG-dT)n sequences

The nucleotide sequences of the introns that are located between the C4 exon and the first membrane exon of mouse and rat immunoglobulin epsilon-chain genes have been determined. The rat intron sequence was found to contain four separate clusters of repetitive sequences all of which consisted of (dC-dA)n.(dG-dT)n dinucleotide repeats. A comparison between this chromosomal region in mouse and rat revealed four deletions or duplications, three of which have occurred inside or at the borders of the CA clusters. Rearrangements have occurred inside or at the borders of all four repeats after the evolutionary separation of mouse and rat. The sequence comparison reveals in addition a duplication, connected to the CA repeats, which has occurred early in evolution, before the evolutionary divergence of mouse and rat. These findings suggest that (dC-dA)n.(dG-dT)n sequences are potential targets for recombination events.     

Molecular and functional organization of yeast plasmid pSR1

The nucleotide sequence of a 6251 base-pair plasmid, pSR1, harbored in an osmophilic haploid yeast, Zygosaccharomyces rouxii (formerly Saccharomyces rouxii), was determined. No homology was detected between the sequences of pSR1 and 2-micron DNA of Saccharomyces cerevisiae. pSR1 has a pair of inverted repeats consisting of completely homologous 959 base-pair sequences, which separate two unique sequences 2654 base-pairs and 1679 base-pairs long. Each inverted repeat has an ARS sequence functional in both Z. rouxii and S. cerevisiae hosts. Short direct repeats or dyad symmetries were observed in the inverted repeats similar to those found close to the replication origin of 2-micron DNA. Three open reading frames, P, S and R, each able to encode a protein of molecular weight larger than 10,000, were found. Insertional inactivation of R gave rise to a defect in the intramolecular recombination at the inverted repeats, and that of S reduced the copy number of pSR1 in the S. cerevisiae host. The maintenance stability of the plasmid was also tested in the heterogeneous S. cerevisiae host, but the results of the insertional inactivation of P, S and R were ambiguous. pSR1 and 2-micron DNA were compatible in S. cerevisiae cells, but the protein factors encoded by these plasmids did not complement each other.     

Identification of a human clustered G + C-rich DNA family of repeats (Sau3A family)

Sau3A digestion of human G + C-rich DNA molecules yields discrete bands of approximately 70 and 140 base-pairs, under-represented in A + T-rich DNA molecules and in total DNA. We have cloned the 70 base-pair band in a plasmid vector and isolated a representative recombinant clone that identifies a new human family of repeats, the Sau3A family. The new family has been characterized for a number of parameters: genomic organization; reiteration frequency; sequence analysis; and distribution in a human genomic library. The Sau3A sequence (68 base-pairs in length, 53% G + C) is present in approximately 4 X 10(4) copies/haploid genome; the family is characterized by a cluster organization and is confined to a limited fraction (0.5%) of phages of a human genomic library. Southern blot hybridizations of the cloned sequence to restriction digests of total human DNA and of isolated genomic clones does not show the involvement of Sau3A blocks in long-range periodicities for any of the enzymes tested. The data suggest either a high sequence variability in the family or a complex organization of Sau3A sequence domains.