Cloning and characterization of two processed pseudogenes and the cDNA for the murine U1 snRNP-specific protein C

Genes for the snRNP proteins U1-70K, U1-A, Sm-B′/B, Sm-D1 and Sm-E have been isolated from various metazoan species. The genes for Sm-D1 and Sm-E, which were isolated from a murine and human source respectively, appear to belong to a multigene family. It has been suggested that also for the mammalian U1-C protein such a multigene family exists. With the human U1-C cDNA as a probe, two genes containing sequences homologous to the probe sequence were isolated from a mouse genomic library. Simultaneously, a murine U1-C cDNA was isolated from a mouse cDNA library. This 0.74 kb cDNA contains an open reading frame (ORF) of 477 bp encoding a polypeptide of 159 amino acids (aa) which differs at only one position (position 65) from the human U1-C protein. One of the isolated U1-C genes contains an ORF as well and shares 92% nucleotide sequence identity with the mouse U1-C cDNA. The features of this gene, in particular the absence of introns, the acquisition of a 3′ poly(A) tail and flanking direct repeats, indicate that it represents a processed pseudogene. At the predicted aa sequence level, substitutions of conserved residues at functionally important positions are observed, strongly suggesting that expression of this gene would not lead to a functional polypeptide. The second U1-C gene appeared to be a pseudogene as well because it is also intronless and contains a frameshift mutation compared to the ORF in the mouse U1-C cDNA. The characterization of these two pseudogenes points to the existence of a U1-C multigene family in mice. Furthermore, comparison of aa sequences of the murine, human and Xenopus U1-C shows that the protein is highly conserved through evolution. Since the Xenopus U1-C differs from the two mammalian counterparts solely at a number of positions in the C-terminal region, it can be concluded that aa changes are less well tolerated in the N-terminal region of U1-C than in the rest of the protein.     

Genomic structure and promoter activity of the testis haploid germ cell-specific intronless genes,Tact1 and Tact2

The Tact1 and Tact2 genes, each of which encodes an actin-like protein, are exclusively expressed and translated in haploid germ cells in testis. To characterize the haploid germ cell-specific gene structure, a mouse genomic library was screened with a Tact1 cDNA as a probe, and four independent phage clones containing the Tact1 gene were isolated. Southern hybridization and sequencing analyses revealed that Tact1 and Tact2 were single copy genes contained on a common fragment in a head-to-head orientation, and that the distance between these genes was less than 2 kb. Comparison of the nucleotide sequences of genomic DNA and cDNA demonstrated that Tact1 and Tact2 lack introns, although all known actin or actin-related genes in mammals contain introns. Human Tact orthologues also lack introns and are located within 6.4 kb in a head-to-head orientation. These findings indicate that Tact1 and Tact2 or one of these genes arose by retroposition of a spliced mRNA transcribed from an actin progenitor gene prior to the divergence of rodents and primates. The Tact1 and Tact2 genes are unusual retroposons in that they have retained an open reading frame and are expressed in testicular germ cells, because almost all retroposons become pseudogenes. It was revealed that a 2kb sequence between the two genes bidirectionally controls haploid germ-cell specific expression by analyzing transgenic mice. Comparison of the murine Tact genes with their human orthologues showed a high level of identity between the two species in the 5'-upstream and non-coding sequences as well as in the coding region, indicating that conserved elements in these regions may be involved in the regulation of haploid germ cell-specific expression. The promoter region contains no TATA-, CCAAT- or GC-boxes, although there are potential cAMP response element (CRE)-like motifs in the 5'-upstream region and the 5'-untranslated region in Tact1 and Tact2, respectively. Transient promoter analyses indicate that CREMtau may activate Tact1 and Tact2 expression in germ cells.     

Structure and hair follicle-specific expression of genes encoding the rat high sulfur protein B2 family

High sulfur proteins are cysteine-rich proteins synthesized during the differentiation of hair matrix cells, and form hair fibers in association with hair keratin intermediate filaments. Rat high sulfur protein B2 genes were isolated after screening of a rat genomic library using the cDNA as a probe. Sequence analysis of a 4 kb fragment revealed two high sulfur protein genes, B2E and B2F. Both genes lacked introns, with B2F being located at 2 kb downstream of B2E. The 5′ flanking regions of both genes had TATA and CAAT boxes, and consensus sequences of B2 genes. The upstream region of B2F had possible AP-1 and Sp-1 binding elements. The high sulfur protein B2E and B2F, which have putative 188 and 122 amino acids, respectively, comprised four distinct domains with a characteristic repetitive sequence. In situ hybridization indicated that the mRNA of high sulfur protein B2 was specifically localized in the cortex of the hair shaft, and Northern blot analysis indicated that the expression of B2 increased in anagen and decreased in telogen, suggesting that high sulfur protein B2 synthesized in cortical cells during anagen contributes to the production of hair fibers.     

T-DNA tagging of the translation initiation factor eIF-4A1 of Arabidopsis thaliana

A transgenic Arabidopsis thaliana line, generated using a T-DNA vector carrying a promoterless gus reporter gene, showed intense GUS expression in young leaves and rapidly growing stem tissues. The gus fusion has tagged the 3′ region of the gene encoding the A. thaliana eukaryotic translation initiation factor eIF-4A1. Comparison of the genomic and cDNA sequence shows that the eIF-4A1 gene contains four introns. Three introns interrupt the translated region, whereas the largest intron splits the 5′-untranslated region. In plants homozygous for the T-DNA markers, the eIF-4A1 and gus genes are expressed as different mature mRNAs. The gus gene is possibly expressed from a cryptic promoter downstream the eIF-4A1 gene.     

Complete nucleotide sequence of a functional HLA-DP beta gene and the region between the DP beta 1 and DP alpha 1 genes: comparison of the 5'' ends of HLA class II genes.

The complete nucleotide sequence of an HLA-DP beta 1 gene and part of the adjacent DP alpha 1 gene, up to and including the signal sequence exon, were determined. The sequence of the DP beta 1 gene identified it as the DPw4 allele. The six exons of the DP beta 1 gene spanned over 11,000 bp of sequence. The arrangement of the gene was broadly analogous to genes of other class II beta chains. The beta 1 exon was flanked by introns of over 4 kb. Comparisons with published sequences of cDNA clones indicated that an alternative splice junction, at the 3' end of the gene, is used in at least one allele. Variation in choice of splice junction indicates an additional mechanism for allelic variation in class II genes. The sequence also indicated that the DP beta 1 and DP alpha 1 genes are separated by only 2 kb at their 5' ends. Comparison of the 5' ends of the DP alpha 1 and beta 1 genes with other class II sequences, including the DZ alpha gene, showed conservation of several blocks of sequences thought to be involved in control of expression. Some areas of the introns were partially conserved in the DQ beta gene, and several other intron sequences were homologous to sequences found in other unrelated genes.     

The chicken H5 gene is unlinked to core and H1 histone genes

An H5 cDNA clone was used to select H5 genomal recombinants from a chicken Charon 4A library. DNA sequence analysis shows that the H5 gene contains no introns. Putative 5′ promoter elements and a 3′ polyadenylation site are present within the 1.8 kb of DNA examined. Analysis of 41 kb of DNA surrounding the H5 gene shows that it is not closely linked to either H1 or core histone genes.     

The plastid aldolase gene fromChlamydomonas reinhardtii: Intron/exon organization,evolution, and promoter structure

Genomic clones encoding the plastidic fructose- 1,6-bisphosphate aldolase ofChlamydomonas reinhardtii were isolated and sequenced. The gene contains three introns which are located within the coding sequence for the mature protein. No introns are located within or near the sequence encoding the transit-peptide, in contrast to the genes for plastidic aldolases of higher plants. Neither the number nor the positions of the three introns of theC. reinhardtii aldolase gene are conserved in the plastidic or cytosolic aldolase genes of higher plants and animals. The 5′ border sequences of introns in the aldolase gene ofC. reinhardtii exhibit the conserved plant consensus sequence. The 3′ acceptor splice sites for introns 1 and 3 show much less similarity to the eukaryotic consensus sequences than do those of intron 2. The plastidic aldolase gene has two tandemly repeated CAAT box motifs in the promoter region. Genomic Southern blots indicate that the gene is encoded by a single locus in theC. reinhardtii genome.     

The structure and expression of maize genes encoding the major heat shock protein, hsp70

We have isolated and sequenced two maize genomic clones that are homologous to the Drosophila hsp70 gene. One of the maize hsp70 clones contains the entire hsp70 coding region and 81 nucleotides of the 5' nontranslated sequence. The predicted amino acid sequence for this maize protein is 68% homologous to the hsp70 of Drosophila. The second maize hsp70 clone contains only part of the coding sequence and 1.1 kb of the 5' flanking sequence. This 5' flanking sequence contains two sequences homologous to the consensus heat-shock-element sequence. Both maize genes are thermally inducible and each contains an intron in the same position as that of the heat-shock-cognate gene, hsc1, of Drosophila. The presence of an intron in the maize genes is a distinguishing feature in that no other thermally inducible hsp70 genes described to date contain an intron. We have constructed a hybrid hsp70 gene containing the entire hsp70 coding sequence with an intron, and 1.1 kb of the 5' flanking sequence. We demonstrate that this hybrid gene is thermally inducible in a transgenic petunia plant and that the gene is expressed from its own promoter.     

Molecular cloning of actin genes in Trichomonas vaginalis and phylogeny inferred from actin sequences

The parasitic protozoan Trichomonas vaginalis is known to contain the ubiquitous and highly conserved protein actin. A genomic library and a cDNA library have been screened to identify and clone the actin gene(s) of T. vaginalis. The nucleotide sequence of one gene and its flanking regions have been determined. The open reading frame encodes a protein of 376 amino acids. The sequence is not interrupted by any introns and the promoter could be represented by a 10 bp motif close to a consensus motif also found upstream of most sequenced T. vaginalis genes. The five different clones isolated from the cDNA library have similar sequences and encode three actin proteins differing only by one or two amino acids. A phylogenetic analysis of 31 actin sequences by distance matrix and parsimony methods, using centractin as outgroup, gives congruent trees with Parabasala branching above Diplomonadida.