The organization of internal telomeric repeats in the polytene chromosomes of the hypotrichous ciliate Stylonychia lemnae.

There exist about 1000-1500 internal telomeric sequences per haploid genome in the polytene chromosomes of the hypotrichous ciliate Stylonychia lemnae. All these telomeric repeats are contained in a very conserved element. This element consists of two 2 kb direct repeats flanking a 2.6 kb sequence. Immediately adjacent to one of the repeats a 18mer C4A4C4A4C2 telomeric sequence is localized. Sequences homologous to macronuclear DNA follow 180 bp downstream of the C4A4-bloc. These macronuclear homologous sequences are flanked by the second direct repeat. The possible origin and function of these telomere containing elements is discussed.     

Membrane and secretory forms of mouse membrane cofactor protein (CD46) generated from a single gene through alternative splicing

 A cDNA encoding a new secretory form of mouse membrane cofactor protein (MCP, CD46) was identified additionally to the membrane form cDNA. The secretory MCP, predicted from the cDNA sequence, consisted of the conserved four short consensus repeats (SCRs) plus a four amino acid-stretch. Unlike human MCP which comprises many isoforms, mouse MCP cDNA predicted a single isoform of membrane MCP with cytoplasmic tail 1 (CYT1) and serine/threonine-rich domain C (ST^C). To clarify the genomic origin and monomorphic alteration of these cDNAs, we cloned and analyzed a mouse genomic DNA harboring the full coding sequence of MCP from a 129/SV mouse genomic library. The mouse Mcp was a single gene ∼50 kilobases long. Eleven of the 14 coding exons of the human MCP gene and intron-exon boundary sequences were found to be conserved in the mouse gene. The ST^C homologue but not the ST^A or ST^B homologue in the mouse exons was functional: the latter being due to deletions and lack of consensus sequences for splicing. The sequence equivalent to cytoplasmic tail 2 (CYT2) has not been identified in the Mcp genome. Thus, the three exons (ST^A, ST^B, and probably CYT2) responsible for the polymorphism of human MCP by differential splicing were missing in the mouse Mcp gene. Unlike the case in humans, no Mcp-related genes or pseudogenes were observed in the mouse genome. The single mouse Mcp gene was mapped to the R-positive H5 band of mouse Chromosome 1 by FISH. Strikingly, one alternative exon with 73 base pairs (encoding the four new amino acids and a TGA stop codon) was discovered between the SCRIV and the ST^C exons; alternative splicing causes the generation of the secretory form of mouse MCP. These results on mouse MCP, together with the information concerning other mouse SCR proteins, infer that the regulator of complement activation (RCA) gene cluster is genetically diverged between humans and mice. Received: 22 April 1999 / Revised: 21 June 1999     

Characterization of a family of repetitive sequences that is eliminated late during macronuclear development of the hypotrichous ciliate Stylonychia lemnae

A repetitive element from the hypotrichous ciliate Stylonychia lemnae was characterized by restriction and hybridization analysis. This repetitive element is present in about 5,000–7,000 copies per haploid genome in the micronucleus and the macronuclear anlagen. Its DNA sequence is very conserved, but the length of the repetitive sequence blocs is variable. In some cases, it is associated with telomeric sequences and macronucleus–homologous sequences. Restriction analysis of genomic micronuclear and macronuclear anlagen DNA and in situ hybridization showed that the repetitive sequences are amplified during the formation of polytene chromosomes. They are localized in many bands of the polytene chromosomes and are eliminated during the degradation of the polytene chromosomes. Possible functions of the repetitive sequences during macronuclear differentiation are discussed. Dev. Genet. 21:201–211, 1997.© 1997 Wiley-Liss, Inc.     

Biased distribution of adenine and thymine in gene nucleotide sequences

We analyzed occurrences of bases in 20,352 introns, exons of 25,574 protein-coding genes, and among the three codon positions in the protein-coding sequences. The nucleotide sequences originated from the whole spectrum of organisms from bacteria to primates. The analysis revealed the following: (1) In most exons, adenine dominates over thymine. In other words, adenine and thymine are distributed in an asymmetric way between the exon and the complementary strand, and the coding sequence is mostly located in the adenine-rich strand. (2) Thymine dominates over adenine not only in the strand complementary to the exon but also in introns. (3) A general bias is further revealed in the distribution of adenine and thymine among the three codon positions in the exons, where adenine dominates over thymine in the second and mainly the first codon position while the reverse holds in the third codon position. The product (A₁/T₁) × (A₂/T₂) × (T₃/A₃) is smaller than one in only a few analyzed genes. Correspondence to: J. Kypr     

C4 genes of the chimpanzee,gorilla, and orang-utan: evidence for extensive homogenization

The human complement component 4 is encoded in two genes, C4A and C4B, residing between the class I and class II genes of the major histocompatibility complex. The C4A and C4B molecules differ in their biological activity, the former binding more efficiently to proteins than to carbohydrates while for the latter, the opposite holds true. To shed light on the origin of the C4 genes we isolated cosmid clones bearing the C4 genes of a chimpanzee, a gorilla, and an orang-utan. From the clones, we isolated the fragments coding for the C4d part of the gene (exons and introns) and sequenced them. Altogether we sequenced eight gene fragments: three chimpanzee (Patr-C4-1 ^*01, Patr-C4-1 ^*02, Patr-C4-2 ^*01), two gorilla (Gogo-C4-1 ^*01, Gogo-C4-2 ^*01), and three orang-utan (Popy-C4-1 ^*01, Popy-C4-2 ^*01, Popy-C4-3 ^*01). Comparison of the sequences with each other and with human C4 sequences revealed that in the region believed to be responsible for the functional difference between the C4A and C4B proteins the C4A genes of the different species fell into one group and the C4B genes fell into another. In the rest of the sequence, however, the C4A and C4B genes of each species resembled each other more than they did C4 genes of other species. These results are interpreted as suggesting extensive homogenization (concerted evolution) of the C4 genes in each species, most likely by repeated unequal, homologous, intragenic crossing-over. Address correspondence and offprint requests to: J. Klein.     

Evolution of the three overlapping gene systems in G4 and phi X174.

Bacteriophage G4 has the same $\text{A}\text{B}$ and $\text{D}\text{E}$ overlapping gene systems as φX174 and together with the A and $\text{C}\text{K}$ overlapping gene system (Shaw et al., 1978), 7 of the 11 G4 and φX174 genes are involved in overlaps. The nucleotide differences between G4 and φX174 in the overlapping portions of the A, C and D genes are 23%, 27% and 21%, respectively, compared with 32%, 36% and 34% in the non-overlapping portions of the same genes. The amino acid differences between the G4 and φX174 overlapping B, K and E proteins, are 44%, 39% and 44%, respectively, compared with 28%, 26% and 16% in the regions of genes A, A and C, and D which contain genes B, K and E. These results suggest that the nucleotide sequences of overlapping genes evolve at almost the same rate as in non-overlapping genes, and that this is made possible by a lower amino acid sequence stringency of one of the pairs of proteins. The overlapping $\text{D}\text{E}$ and A and $\text{C}\text{K}$ gene systems may have originated by taking advantage of a high incidence of T nucleotides in the second codon position to produce a hydrophobic protein and the $\text{A}\text{B}$ gene system may have evolved by read-through of the A gene into the B gene. From the nucleotide sequences, other overlapping genes appear to be possible in these bacteriophages.     

Structure of Amylase Genes in Populations of Pacific Cupped Oyster (Crassostrea gigas): Tissue Expression and Allelic Polymorphism

Using the previously determined complementary DNA Sequence of Crassostrea gigas amylase (Y08370), we designed several oligonucleotide primers and used them with polymerase chain reaction (PCR) technology to characterize oyster amylase gene sequences. Two genes encoding 2 different amylases were characterized and sequenced. The 2 genes are similarly organized with 8 exons and 7 introns. Intron insertions are found at the same location in the 2 genes. Sizes and nucleotide sequences are different for the different introns inside each gene and different for the corresponding introns in the 2 genes. Comparing the 2 genes, around 10% of the nucleotides are different along the exons, and comparing the 2 deduced protein sequences, a mean value of 10.4% of amino acids are changed. Genes A and B encode mature proteins of, respectively, 500 and 499 amino acids, which present 94% similarity. A microsatellite (TC₃₇) that constitutes the largest part of intron 4 of gene A has been used as a polymorphic marker. A method consisting of a PCR step followed by EcoRI digestion of the obtained fragments was used to observe polymorphism in these 2 genes. Six and 4 alleles for genes A and B, respectively, have been sequenced, leading to a maximum of 2.9% base change. The 2 genes are ubiquitously expressed in the different digestive tissues with quantitative differences. Gene A is strongly expressed in the digestive gland and at a lower level in stomach, while gene B is preferentially expressed in the labial palps. The microsatellite repeat was used in the analysis of 4 populations of Crassostrea gigas from the French Atlantic coast. A high level of polymorphism observed with 30 different alleles of gene A inside the populations should allow their characterization using the mean value of the microsatellite allelic distribution. These populations showed a low level of differentiation (F _st between 0 and 0.011); however, the population of Bonne Anse appeared to be distinguished from the other populations.     

The Micronuclear Gene Encoding β-Telomere Binding Protein in Oxytricha nova

ABSTRACT The micronuclear version of the gene encoding β-telomere binding protein (β-TBP) in Oxytricha nova has been sequenced and compared to the macronuclear β-TBP gene, previously described. The micronuclear gene contains three AT-rich internal eliminated sequences (IES) of 37, 40, and 43 bp and four macronuclear destined sequences (MDS). The IES interrupt the gene once near the 5′ end of the coding region and twice in the 3′ trailer downstream from the TGA stop codon. The sequences of the micronuclear and macronuclear genes are colinear. Thus, the micronuclear β-TBP gene is not scrambled, which contrasts with the highly scrambled state among the 14 MDS in the micronuclear α;-TBP gene.     

A new repetitive sequence uniquely present in the decay-accelerating factor genes

 The decay-accelerating factor (DAF, CD55) protects cells from autologous complement attack on self cell membranes. We have previously reported that the seventh exon encoding the serine/threonine-rich(S/T)-abc region of the guinea pig DAF gene is composed of five homologous repeats of about 51 base pairs, and that differential usage of these repeats produces the various lengths observed in the S/T region of guinea pig DAF. In this study, we found that the seventh intron of the guinea pig DAF gene was wholly composed of 18 tandem repeats homologous to the repeating unit of the S/T-abc exon. This type of repetitive structure, although the number of repeats was variable, was also found in the corresponding exons and introns of all DAF genes of other species so far tested including human and seven other primates and mouse, in which alternative splicing in this region has not been found. This suggested that generation of the repetitive sequences spanning the exon and intron regions had occurred before the diversification of these species. In addition, all the intron sequences of the tested DAF genes had no stop codon when they were presumably translated in the same reading frame as the seventh and eighth exons, except for that of one of two duplicated mouse DAF genes. These findings and significant interspecies identities of the intron sequence suggest that the intron sequence conceivably could be translated in some tissues and/or in some stages of development although to date we have not yet succeeded in detecting mRNA for this region. Received: 7 July 1997 / Revised: 11 August 1997     

The nucleotide sequence of the bovine MHC class II alpha genes:DRA,DQA, andDYA

The nucleotide sequence of the exons 2, 3, and 4, and parts of the intervening sequences of aBoLA-DRA and-DQA gene and one other class IIBoLA-A gene have been determined. The structure of theBoLA-DRA and-DQA gene was found to be very similar to that of the corresponding human HLA class II genes. An analysis of the structure of the other class IIBoLA-A gene showed that thisA gene was clearly very different from both the humanA genes and the bovineDRA andDQA genes. The results indicate that this other type of class IIA gene probably represents the class II gene that has already been identified in restriction fragment length polymorphism (RFLP) studies asBoLA-DYA. Since no clear homologue of this presumedBoLA-DYA gene was found among the human HLA class II genes, these results indicate that, at least as far as theA genes are concerned, a distinct class II gene is present 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 M30117–M30120. Address correspondence and offprint requests to: J. van der Poel.     

Conserved linkage of two genes on the same macronuclear chromosome in spirotrichous ciliates

Macronuclear chromosomes of spirotrichous ciliates are mainly “nanochromosomes” containing only a single gene. We identified a two-gene chromosome in the spirotrich Sterkiella histriomuscorum (formerly Oxytricha trifallax) which, unlike other characterized two-gene molecules, contains reading frames oriented tail to tail. These are homologs of ribosomal protein L29 (RPL29) and cyclophilin. We found that both genes are transcribed, with their polyadenylation sites on opposite strands separated by only 135 bp. Furthermore, both genes in S. histriomuscorum are present only on one macronuclear chromosome and do not occur alone or linked to other genes. The corresponding micronuclear locus is fragmented into three nonscrambled gene segments (MDSs), separated by two noncoding segments (IESs). We also found that these two genes are linked on a macronuclear chromosome, similarly arranged tail to tail, in the three spirotrichs Stylonychia lemnae, Uroleptus sp., and Holosticha sp.. In addition, single-gene macronuclear chromosomes containing only the RPL29 gene were detected in the earlier diverged Holosticha and Uroleptus. These observations suggest a possible evolutionary trend towards loss of chromosomal breakage between these two genes. This study is the first to examine gene linkage in the macronucleus of several spirotrichs and may provide insight into the evolution of multi-gene macronuclear chromosomes and chromosomal fragmentation in spirotrichs. Electronic Supplementary Material Supplementary material is available for this article at     

Contrasting response of euchromatin and heterochromatin to translocation in polytene chromosomes of Drosophila melanogaster

Studies on Feulgen-DNA content in the polytene chromosomes of D. melanogaster T(14)w ^m258-21 heterozygotes showed that when the euchromatic region 3D₁-E₂ is located next to the heterochromatic breakpoint it contains less DNA than in the non-translocated homologue (Hartmann-Goldstein and Cowell, 1976). In contrast to the region adjacent to the breakpoint, region 3C_1–10, which contains intercalary heterochromatin, shows more DNA in the translocated than in the non-translocated chromosome. Transposition may induce morphologically euchromatic regions containing putatively underreplicated sequences to undergo additional replication cycles. Region 2E₁-3A₄, distal to 3C₁ and at some distance from the heterochromatic breakpoint is apparently unaffected. Extended replication and reduced DNA content in regions which have undergone chromosomal rearrangement could be accounted for by varying degrees of blockage of replication in individual strands of the polytene chromosome.     

Timing of differential amplification of macronucleus-destined sequences during macronuclear development in the hypotrichous ciliateEuplotes crassus

The change in copy numbers of macronucleus-destined gene sequences was followed in anlagen DNA during postconjugational development inEuplotes crassus. As noted earlier, copy numbers increase during the polytene stage. During this replication process major differential amplification of different genes is not observed. Instead it is only achieved during or shortly after the fragmentation of the polytene chromosomes. This process is not totally synchronous with respect to different genes. Highly amplified genes are excised earlier than genes with a low final macronuclear copy number. Unexpectedly, the pattern of processing of the newly added oversized telomeres also appears to correlate with the degree of gene amplification. These observations are discussed in terms of a limited replication period after polytene chromosome fragmentation leading to preferential amplification of early excised genes.     

Genomic characterization and linkage mapping of the apple allergen genes Mal d 2 (thaumatin-like protein) and Mal d 4 (profilin)

Four classes of apple allergens (Mal d 1, −2, −3 and −4) have been reported. By using PCR cloning and sequencing approaches, we obtained genomic sequences of Mal d 2 (thaumatin-like protein) and Mal d 4 (profilin) from the cvs Prima and Fiesta, the two parents of a European reference mapping population. Two copies of the Mal d 2 gene (Mal d 2.01A and Mal d 2.01B) were identified, which primarily differed in the length of a single intron (378 or 380 nt) and in one amino acid in the signal peptide. Both Mal d 2.01A and Mal d 2.01B were mapped at identical position on linkage group 9. Genomic characterization of four Mal d 4 genes (Mal d 4.01A and B, Mal d 4.02A and Mal d 4.03A) revealed their complete gDNA sequences which varied among genes in length from 862 to 2017 nt. They all contained three exons of conserved length: 123, 138, and 135 nt. Mal d 4.01 appeared to be duplicated in two copies and located on linkage group 9. Mal d 4.02A and Mal d 4.03A were single copy genes located on linkage group 2 and 8, respectively.