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PubMed1998

Structure and expression of the gene encoding murine M-protein, a sarcomere-specific member of the immunoglobulin superfamily.

The complete exon-intron organization of the murine gene encoding M-protein, a structural protein of sarcomeric myofibrils, was determined. The gene is composed of 37 exons and 36 introns, spanning approximately 75 kb of DNA. Intron positions are related to the modular structure of M-protein, which is composed essentially of immunoglobulin and fibronectin type III domains. Almost all repeats follow a two exon-one domain structure. The beginning and end of each domain are defined by introns in phase I; internal introns are more divergent in position and very rarely use phase I. A single transcriptional start point was detected in both skeletal and cardiac muscle. Analysis of the prospective promoter region revealed several potential regulatory elements. CAT expression assays using promoter deletion constructs identified three regions that seem to be most important for the muscle-specific transcription activation of the M-protein gene. These results provide the first complete characterization of a gene for a member of the intracellular branch of the immunoglobulin superfamily.

Amino Acid Sequence↗
PubMed1993

A splice site mutation of alpha-spectrin gene causing skipping of exon 18 in hereditary elliptocytosis.

Spectrin Oran (alpha II/21) has been reported previously as a variant of the alpha II domain. Its expression level is low (10% of total spectrin) in heterozygotes denoting a major disadvantage of the mutated alpha-chain dimer or tetramer with respect to their normal counterparts. Spectrin Oran is associated with symptomatic elliptocytosis in the homozygous state. A 1-minute digestion time allowed to perceive a fast trypsin cleavage (not existing normally) after Arg 890 (helix 3 of repeating segment alpha 9). The responsible change was the lack of amino acids 822 to 862 (helix 2 of repeating segment alpha 8). Such a situation fits with the phasing of spectrin according to which mutated helix 2 and distorted helix 3 are adjacent to one another. The internal position of the structural change accounts for the slight self-association defect. The ultimate genetic lesion was a G to A substitution (intronic position-1) in the acceptor splice site of intron 17 resulting in skipping of exon 18. The substitution also created an acceptor splice site 1 base downstream, but the latter was used at a low grade.

Alleles↗
PubMed1994

Differential expression of multiple exo-cellobiohydrolase I-like genes in the lignin-degrading fungus Phanerochaete chrysosporium.

The genome of Phanerochaete chrysosporium strain ME446 contains multiple, non-allelic, cellobiohydrolase I (CBHI)-like sequences, at least two of which are expressed in a cellulose-dependent manner. Each of the expressed genes contains two identically positioned introns within its coding region. The lengths and sequences of these introns are different and one is not excised from all transcripts, raising the possibility that subtly different protein products may be expressed from a common gene. Introns are also present upstream of both genes but these differ in number and position, as well as sequence and length. Endoglucanase-like sequences could not be identified and it is suggested that variant CBHI-like proteins may provide endoglucanase activity in this fungus.

Amino Acid Sequence↗
PubMed1996

Structural characterization of the mouse ribosomal protein S6-encoding gene.

The gene encoding mouse ribosomal protein (r-protein) S6 is 2.7 kb in length, and is composed of five exons. The intron positions of the mouse S6 (Rps6) coincide exactly to those of the homologous human S6 (RPS6), but the last intron present in the human is absent in the mouse gene. The latter displays higher G + C content than the RPS6, both in the overall sequenced region and at the 3rd codon position. The promoter area is highly conserved between mouse and human, and contains several putative cis-acting elements. Comparison of the intronic sequences of both genes revealed surprisingly a high degree of identity (63%) within 350 bp of the first intron. Besides the single-copy Rsp6 there are up to 15 S6 family members, most likely processed pseudogenes. Characterization of the Rps6 provides a basis to study the functions of the mammalian S6 by gene targeting.

Amino Acid Sequence↗
PubMed1994

Structure of the gene coding for the human early lymphocyte activation antigen CD69: a C-type lectin receptor evolutionarily related with the gene families of natural killer cell-specific receptors.

CD69 is the earliest inducible cell surface glycoprotein acquired during lymphoid activation. CD69 functions as a signal transmitting receptor involved in cellular activation events including proliferation and the induction of specific genes. This molecule is a member of a supergene family of type-II integral membrane proteins with C-type lectin domains. We have herein studied the genomic structure of the human gene encoding CD69. The coding sequence is divided into five exons separated by four introns. The first two exons corresponded to separate functional domains of the protein (cytoplasmic tail and the transmembrane region), while the final three exons encoded the carbohydrate-recognition domain (CRD). The conserved intron position between the exons encoding the CRD indicated that this protein is closely related to other type-II receptor groups with the C-type CRD, such as the asialoglycoprotein receptors, the low-affinity IgE receptor (CD23), and natural killer cell-specific receptors, NKR-P1 and Ly49. In contrast to the broad NKR-P1 and Ly-49 gene families, CD69 is a single-copy gene, as demonstrated by Southern blot analyses. The major transcription initiation site has been located, by amplification of cDNA 5' ends, 30 nucleotides downstream of a consensus TATA box. Comparison of human CD69 and mouse NKR-P1 gene structures indicates that the first intron maintains a conserved position, suggesting that CD69 and this gene family may diverge from a common ancestor gene. A possible evolutionary pathway of these genes is proposed.

Antigens, CD↗
PubMed1999

M band proteins myomesin and skelemin are encoded by the same gene: analysis of its organization and expression.

The complete exon-intron organization of the murine gene encoding sarcomeric myomesin has been determined. The gene is composed of 38 exons and 37 introns, spanning approximately 105 kb of DNA. Intron positions and phases are essentially identical to those identified in M-protein. They are related to the modular structure of myomesin, which is composed almost entirely of immunoglobulin and fibronectin type III domains. Nearly all repeats follow a two exon-one domain structure. The start and end of each domain are defined by introns in phase I, while internal introns are more divergent in position and very rarely use phase I. Genomic Southern blotting and reverse transcription-polymerase chain reaction revealed that differential splicing of a single exon gives rise to two polypeptides, described in the literature as myomesin and skelemin, respectively. A single transcriptional start point was detected in both skeletal and cardiac muscle. Analysis of the presumptive promoter region revealed several potential regulatory elements. CAT expression assays using promoter deletion constructs identified three regions that seem to be important for the muscle-specific transcriptional activation of the myomesin gene. These results provide the basis for a comparative analysis of the regulation of myomesin and M-protein genes in vivo.

Alternative Splicing↗
PubMed1998

A combined analysis of genomic and primary protein structure defines the phylogenetic relationship of new members if the T-box family.

T-box genes form an ancient family of putative transcriptional regulators characterized by a region of homology to the DNA-binding domain of the murine Brachyury (T) gene product. This T-box domain is conserved from Caenorhabditis elegans to human, and mutations in T-box genes have been associated with developmental defects in Drosophila, zebrafish, mice, and humans. Here we report the identification of three novel murine T-box genes and an investigation of their evolutionary relationship to previously known family members by studying the genomic structure of the T-box. All T-box genes from nematodes to humans possess a characteristic central intron that presumably was inherited from a common ancestral precursor. Two additional intron positions are also conserved with the exception of two nematode T-box genes. Subsequent intron insertions, potential deletions, and/or intron sliding formed a structural basis for the divergence into distinct subfamilies and a substrate for length variations of the T-box domain. In mice, the 11 T-box genes known to date can be grouped into seven subfamilies. Genes assigned to the same subfamily by genomic structure show related expression patterns. We propose a model for the phylogenetic relationships within the gene family that provides a rationale for classifying new T-box genes and facilitates interspecific comparisons.

Amino Acid Sequence↗
PubMed1987

Structure and organization of the CyIII actin gene subfamily of the sea urchin, Strongylocentrotus purpuratus.

We describe here the organization of the CyIII subfamily of cytoskeletal actin genes in the sea urchin Strongylocentrotus purpuratus. The functional genes CyIIIa and CyIIIb are linked at a 6 X 10(3)-base distance. Gene CyIIIc appears to be a pseudogene that lacks 5' exons and displays unselected mutational changes. Gene CyIIIa codes for a protein that differs at only nine out of 376 residues from that coded by another cytoskeletal actin gene, CyI. However, five of these nine changes occur within an 11-amino acid region that could represent a functional specialization of the CyIIIa actin protein. The CyIIIa gene possesses three introns, located, respectively, 25 nucleotides upstream from the translation start site, between the codons for amino acids 121 and 122, and within the codon for amino acid 204. These intron positions have also been observed in other cytoskeletal sea urchin actin genes. Comparison of both intron and 3'-terminal sequences shows that the CyIIIa and CyIIIb genes are closely related, while no homology in these untranslated sequences is observed between the CyIII genes and the other cytoskeletal actin genes of the S. purpuratus genome. The CyIII genes probably arose by duplication events at least 40 X 10(6) years ago, prior to radiation of the genus Strongylocentrotus. Consideration of the biological role of the embryo and larval aboral ectoderm cells to which CyIIIa and CyIIIb transcripts are confined suggests that these actins might contribute to cytoskeletal elements that endow the larval body wall with its rigid structure.

Actins↗
PubMed1997

Subunits I and II of Dictyostelium cytochrome c oxidase are specified by a single open reading frame transcribed into a large polycistronic RNA.

A single open reading frame (ORF) encoding cytochrome c oxidase subunit I and II (cox1/2) was identified in the mitochondrial genome of the slime mold Dictyostelium discoideum. The cox1 coding region shares intron positions with its counterparts in fungi and algae. Northern blot analysis, using exon and intron-specific probes, suggests that the cox1/2 gene is transcribed as part of a large, efficiently processed, polycistronic RNA.

Amino Acid Sequence↗
PubMed2004

Comparative analysis detects dependencies among the 5' splice-site positions.

Human-mouse comparative genomics is an informative tool to assess sequence functionality as inferred from its conservation level. We used this approach to examine dependency among different positions of the 5' splice site. We compiled a data set of 50,493 homologous human-mouse internal exons and analyzed the frequency of changes among different positions of homologous human-mouse 5' splice-site pairs. We found mutual relationships between positions +4 and +5, +5 and +6, -2 and +5, and -1 and +5. We also demonstrated the association between the exonic and the intronic positions of the 5' splice site, in which a stronger interaction of U1 snRNA and the intronic portion of the 5' splice site compensates for weak interaction of U1 snRNA and the exonic portion of the 5' splice site, and vice versa. By using an ex vivo system that mimics the effect of mutation in the 5' splice site leading to familial dysautonomia, we demonstrated that U1 snRNA base-pairing with positions +6 and -1 is the only functional requirement for mRNA splicing of this 5' splice site. Our findings indicate the importance of U1 snRNA base-pairing to the exonic portion of the 5' splice site.

Animals↗
PubMed2002

The chaperonin genes of jakobid and jakobid-like flagellates: implications for eukaryotic evolution.

The jakobids are free-living mitochondriate protists that share ultrastructural features with certain amitochondriate groups and possess the most bacterial-like mitochondrial genomes described thus far. Jakobids belong to a diverse group of mitochondriate and amitochondriate eukaryotes, the excavate taxa. The relationships among the various excavate taxa and their relationships to other putative deep-branching protist groups are largely unknown. With the hope of clarifying these issues, we have isolated the cytosolic chaperonin CCTalpha gene from the jakobid Reclinomonas americana (strains 50394 and 50283), the jakobid-like malawimonad Malawimonas jakobiformis, two heteroloboseans (Acrasis rosea and Naegleria gruberi), a euglenozoan (Trypanosoma brucei), and a parabasalid (Monocercomonas sp.). We also amplified the CCTdelta gene from M. jakobiformis. The Reclinomonas and Malawimonas sequences presented here are among the first nuclear protein-coding genes to be described from these organisms. Unlike other putative early diverging protist lineages, a high density of spliceosomal introns was found in the jakobid and malawimonad CCTs-similar to that observed in vertebrate protein-coding genes. An analysis of intron positions in CCT genes from protists, plants, animals, and fungi suggests that many of the intron-sparse or intron-lacking protist lineages may not be primitively so but have lost spliceosomal introns during their evolutionary history. In phylogenetic trees constructed from CCTalpha protein sequences, R. americana (but not M. jakobiformis) shows a weak but consistent affinity for the Heterolobosea and Euglenozoa.

Animals↗
PubMed1998

Genetic interaction between U6 snRNA and the first intron nucleotide in Saccharomyces cerevisiae.

Nuclear pre-mRNA splicing necessitates specific recognition of the pre-mRNA splice sites. It is known that 5' splice site selection requires base pairing of U6 snRNA with intron positions 4-6. However, no factor recognizing the highly conserved 5' splice site GU has yet been identified. We have tested if the known U6 snRNA-pre-mRNA interaction could be extended to include the first intron nucleotides and the conserved 50GAG52 sequence of U6 snRNA. We observe that some combinations of 5' splice site and U6 snRNA mutations produce a specific synthetic block to the first splicing step. In addition, the U6-G52U allele can switch between two competing 5' splice sites harboring different nucleotides following the cleavage site. These results indicate that U6 snRNA position 52 interacts with the first nucleotide of the intron before 5' splice site cleavage. Some combinations of U6 snRNA and pre-mRNA mutations also blocked the second splicing step, suggesting a role for the corresponding nucleotides in a proofreading step before exon ligation. From studies in diverse organisms, various functions have been ascribed to the conserved U6 snRNA 47ACAGAG52 sequence. Our results suggest that these discrepancies might reflect variations between different experimental systems and point to an important conserved role of this sequence in the splicing reaction.

Base Sequence↗
PubMed1987

Mutations at the lariat acceptor site allow self-splicing of a group II intron without lariat formation.

The fifth intron in the gene for cytochrome c oxidase subunit I in yeast mitochondrial DNA is of the group II type and is capable of self-splicing in vitro. The reaction results in lariat formation, concomitant with exon-exon ligation and does not require a guanosine nucleotide for its initiation. It is generally assumed, but not formally proven, that the first step in splicing is a nucleophilic attack of the 2'-hydroxyl of the branchpoint nucleotide (A) on the 5'-exon-intron junction. To investigate the role of intron sequences in recognition of the 5'-splice junction and the ensuing event of cleavage and lariat formation, mutations have been introduced at and around the branchsite. Results obtained show that although branchpoint attack and subsequent lariat formation are strongly preferred events under conditions normally used for self-splicing, addition of a single T residue at intron position 856, a mutation which brings the branchpoint adenosine into a basepair, leads to a conditionally active intron, which at high ionic strength catalyses exon-exon ligation in the absence of lariat formation. Comparable behaviour is also observed with the branchpoint A deletion mutant. The implications of these findings for the mechanism of self-splicing of group II introns are discussed.

DNA, Mitochondrial↗
PubMed2001

Evolution of intron/exon structure of DEAD helicase family genes in Arabidopsis, Caenorhabditis, and Drosophila.

The DEAD box RNA helicase (RH) proteins are homologs involved in diverse cellular functions in all of the organisms from prokaryotes to eukaryotes. Nevertheless, there is a lack of conservation in the splicing pattern in the 53 Arabidopsis thaliana (AtRHs), the 32 Caenorhabditis elegans (CeRHs) and the 29 Drosophila melanogaster (DmRHs) genes. Of the 153 different observed intron positions, 4 are conserved between AtRHs, CeRHs, and DmRHs, and one position is also found in RHs from yeast and human. Of the 27 different AtRH structures with introns, 20 have at least one predicted ancient intron in the regions coding for the catalytic domain. In all of the organisms examined, we found at least one gene with most of its intron predicted to be ancient. In A. thaliana, the large diversity in RH structures suggests that duplications of the ancestral RH were followed by a high number of intron deletions and additions. The very high bias toward phase 0 introns is in favor of intron addition, preferentially in phase 0. Results from this comparative study of the same gene family in a plant and in two animals are discussed in terms of the general mechanisms of gene family evolution.

Animals↗
PubMed2005

SSCP-SNP in pearl millet--a new marker system for comparative genetics.

A considerable array of genomic resources are in place in pearl millet, and marker-aided selection is already in use in the public breeding programme at ICRISAT. This paper describes experiments to extend these publicly available resources to a single nucleotide polymorphism (SNP)-based marker system. A new marker system, single-strand conformational polymorphism (SSCP)-SNP, was developed using annotated rice genomic sequences to initially predict the intron-exon borders in millet expressed sequence tags (ESTs) and then to design primers that would amplify across the introns. An adequate supply of millet ESTs was available for us to identify 299 homologues of single-copy rice genes in which the intron positions could be precisely predicted. PCR primers were then designed to amplify approximately 500-bp genomic fragments containing introns. Analysis of these fragments on SSCP gels revealed considerable polymorphism. A detailed DNA sequence analysis of variation at four of the SSCP-SNP loci over a panel of eight inbred genotypes showed complex patterns of variation, with about one SNP or indel (insertion-deletion) every 59 bp in the introns, but considerably fewer in the exons. About two-thirds of the variation was derived from SNPs and one-third from indels. Most haplotypes were detected by SSCP. As a marker system, SSCP-SNP has lower development costs than simple sequence repeats (SSRs), because much of the work is in silico, and similar deployment costs and through-put potential. The rates of polymorphism were lower but useable, with a mean PIC of 0.49 relative to 0.72 for SSRs in our eight inbred genotype panel screen. The major advantage of the system is in comparative applications. Syntenic information can be used to target SSCP-SNP markers to specific chromosomal regions or, conversely, SSCP-SNP markers can be used to unravel detailed syntenic relationships in specific parts of the genome. Finally, a preliminary analysis showed that the millet SSCP-SNP primers amplified in other cereals with a success rate of about 50%. There is also considerable potential to promote SSCP-SNP to a COS (conserved orthologous set) marker system for application across species by more specifically designing primers to precisely match the model genome sequence.

Base Sequence↗
PubMed1995

Protein L-isoaspartyl methyltransferase from the nematode Caenorhabditis elegans: genomic structure and substrate specificity.

We identified a protein L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) in the nematode worm Caenorhabditis elegans. The methylation of abnormal L-isoaspartyl residues by this enzyme can lead to their conversion to L-aspartyl residues and represents a protein repair step for polypeptides damaged by spontaneous reactions during the aging process. We show that the levels of this enzyme increase 2-fold in C. elegans in the dauer larval form, a developmental stage where the organism can survive for extended periods of time. Utilizing degenerate oligonucleotide primers derived from conserved amino acid sequences of mammalian, plant, and bacterial L-isoaspartyl methyltransferases and PCR amplification, we made DNA probes that allowed us to obtain cDNA and genomic DNA clones encoding this enzyme in the nematode. The deduced amino acid sequence is 53% identical to the human enzyme and 29% identical to the Escherichia coli enzyme. Overexpression of the cDNA for the C. elegans enzyme in E. coli gave an active product with micromolar Km values for L-isoaspartyl-containing peptide substrates and for the methyl donor S-adenosyl-L-methionine. No methylation of D-aspartyl-containing peptides was detected under conditions where the human enzyme catalyzed this reaction, suggesting that the ability to methylate D-aspartyl residues in addition to L-isoaspartyl residues was a later evolutionary adaptation of this enzyme. The C. elegans gene for the methyltransferase, designated pcm-1, was mapped to a single site in a 31 kb region in the central portion of chromosome V. The gene is 3.2 kb in length and includes six introns. Although much smaller, its genomic organization is similar to that of the corresponding mouse gene, with identically positioned intron--exon splice junctions at five of seven sites. We propose that this gene plays an important role in facilitating the long term survival of this organisms.

Amino Acid Sequence↗
PubMed2006

The evolution of spliceosomal introns: patterns, puzzles and progress.

The origins and importance of spliceosomal introns comprise one of the longest-abiding mysteries of molecular evolution. Considerable debate remains over several aspects of the evolution of spliceosomal introns, including the timing of intron origin and proliferation, the mechanisms by which introns are lost and gained, and the forces that have shaped intron evolution. Recent important progress has been made in each of these areas. Patterns of intron-position correspondence between widely diverged eukaryotic species have provided insights into the origins of the vast differences in intron number between eukaryotic species, and studies of specific cases of intron loss and gain have led to progress in understanding the underlying molecular mechanisms and the forces that control intron evolution.

Animals↗
PubMed2004

Intron size correlates positively with recombination rate in Caenorhabditis elegans.

A negative correlation between intron size and recombination rate has been reported for the Drosophila melanogaster and human genomes. Population-genetic models suggest that this pattern could be caused by an interaction between recombination rate and the efficacy of natural selection. To test this idea, we examined variation in intron size and recombination rate across the genome of the nematode Caenorhabditis elegans. Interestingly, we found that intron size correlated positively with recombination rate in this species.

Animals↗