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PubMed1998

Isolation and identification of novel protein kinase genes from the round-spotted pufferfish (Tetraodon fluviatilis) genomic DNA.

The round-spotted pufferfish Tetraodon fluviatilis has a genome size of 380 Mb which is slightly smaller than that of another pufferfish, Fugu rubripes rubripes (Fugu). Due to their compact genome and small introns, both pufferfishes have been proposed as model organisms for genome studies. In this study, we have used genomic DNA as template to perform PCR to screen for protein kinase (pk) genes. Forty-one T. fluviatilis pk genes encoding 7 receptor tyrosine kinases, 14 nonreceptor tyrosine kinases, 16 serine/threonine kinases, 1 dual kinase and 3 novel kinases have been identified. The success of this approach depends on the size and location of the introns. Most of the identified pk gene fragments contain introns, ranging from 71 to 300 bp, with an average of 120 bp. It is noteworthy that the intron/exon boundaries of certain genes which belong to the same family are identical. We also analyzed by specific RT-PCR primers the expression profile of those 3 novel genes as well as some selected pk genes in a variety of tissues. We found that erbB3, pku a, mrk, CaMK I, CaMKIIgamma, and two novel kinase genes (133 and 3-26) are expressed in all tissues examined. However, the novel clone 146 is strongly expressed in the brain and weakly in the intestine, kidney and heart.

Amino Acid Sequence↗
PubMed1998

Round-spotted pufferfish (Tetraodon fluviatilis) snf5 gene is oriented in a tail-to-tail manner with the set gene which encodes an inhibitor of protein phosphatase 2A.

The round-spotted pufferfish Tetraodon fluviatilis has a genome size of 380 Mb which is slightly smaller than that of another pufferfish Fugu rubripes rubripes (Fugu). Due to its compact genome and small introns, Fugu has been introduced as a model for genome studies. Recently, the round-spotted pufferfish has also been proposed as a new model for genome studies because of the ease in obtaining material and high-sequence homology to that of Fugu. In this study, we have cloned and characterized the snf5 and set genes from the round-spotted pufferfish. The snf5 gene is composed of 9 exons spanning about 2.9 kb whereas the set gene consists of 8 exons spanning about 2.7 kb. They are linked in a tail-to-tail manner with an intergenic region of about 6.5 kb. So far, the genomic structures of human snf5 and set genes are unknown. Based on our data, the pufferfish SNF5 and SET display high amino acid sequence identity (>90%) with the respective human genes. By primer extension and sequence analysis, we found that putative promoter region of the snf5 gene contains a typical TATA box and numerous potential binding sites for transcription factors including AP1, AP2, AP3, c-Myb, HNF-5, and NF-IL6. As for the set gene, its promoter region does not have any TATA or CCAAT motif and contains a few potential binding sites for transcriptional factors such as c-Myb and gamma-IRE. When these promoter regions were placed upstream of the CAT reporter gene and transfected into a carp CF cell line, the 5'-upstream 1.6-kb DNA fragment of the snf5 gene displayed stronger promoter activity, approximately three-fold higher than that of the 5'-upstream 1.3 kb DNA fragment of the set gene. By transient expression and immunofluorescent staining, we also showed that the pufferfish SNF5 and SET are nuclear proteins, consistent with their postulated roles as transcriptional factors.

Amino Acid Sequence↗
PubMed2004

The regulatory content of intergenic DNA shapes genome architecture.

BACKGROUND: Factors affecting the organization and spacing of functionally unrelated genes in metazoan genomes are not well understood. Because of the vast size of a typical metazoan genome compared to known regulatory and protein-coding regions, functional DNA is generally considered to have a negligible impact on gene spacing and genome organization. In particular, it has been impossible to estimate the global impact, if any, of regulatory elements on genome architecture. RESULTS: To investigate this, we examined the relationship between regulatory complexity and gene spacing in Caenorhabditis elegans and Drosophila melanogaster. We found that gene density directly reflects local regulatory complexity, such that the amount of noncoding DNA between a gene and its nearest neighbors correlates positively with that gene's regulatory complexity. Genes with complex functions are flanked by significantly more noncoding DNA than genes with simple or housekeeping functions. Genes of low regulatory complexity are associated with approximately the same amount of noncoding DNA in D. melanogaster and C. elegans, while loci of high regulatory complexity are significantly larger in the more complex animal. Complex genes in C. elegans have larger 5' than 3' noncoding intervals, whereas those in D. melanogaster have roughly equivalent 5' and 3' noncoding intervals. CONCLUSIONS: Intergenic distance, and hence genome architecture, is highly nonrandom. Rather, it is shaped by regulatory information contained in noncoding DNA. Our findings suggest that in compact genomes, the species-specific loss of nonfunctional DNA reveals a landscape of regulatory information by leaving a profile of functional DNA in its wake.

Animals↗
PubMed1997

The comparative genomic structure and sequence of the surfeit gene homologs in the puffer fish Fugu rubripes and their association with CpG-rich islands.

The puffer fish Fugu rubripes (Fugu) has a compact genome approximately one-seventh the size of man, mainly owing to small intron size and the presence of few dispersed repetitive DNA elements, which greatly facilitates the study of its genes at the genomic level. It has been shown previously that, whereas the Surfeit genes are tightly clustered at a single locus in mammals and birds, the genes are found at three separate loci in the Fugu genome. Here, Fugu gene homologs of all six Surfeit genes (Surf-1 to Surf-6) have been cloned and sequenced, and their gene structure has been compared with that of their mammalian and avian homologs. The predicted protein products of each gene are well conserved between vertebrate species, and in most cases their gene structures are identical to their mammalian and avian homologs except for the Fugu Surf-6 gene, which was found to lack an intron present in the mouse gene. In addition, we have identified conserved regulatory elements at the 5' and 3' ends of the Surf-3/rpL7a gene by comparison with the mammalian and chicken Surf-3/rpL7a gene homologs, including the presence of a polypyrimidine tract at the extreme 5' end of this ribosomal protein gene. The Fugu Surfeit gene homologs appear to be associated with CpG-rich islands, like the Surfeit genes in higher vertebrates, but these Fugu CpG islands are similar to the nonclassical islands characteristic of other fish species. Our observations support the use of the Fugu genome to study vertebrate gene structure, to predict the structure of mammalian genes, and to identify vertebrate regulatory elements. [The sequence data described in this paper have been submitted to the data library under accession nos. Y15170 (Surf-2, Surf-4), Y15171 (Surf-3, Surf-1, Surf-6), and Y15172 (Surf-5.)]

Amino Acid Sequence↗
PubMed2006

A novel primate specific gene, CEI, is located in the homeobox gene IRXA2 promoter in Homo sapiens.

The Iroquois (IRX) homeobox gene family consists of six highly conserved transcription factors that are of importance for normal embryonic development. They are organized in two gene clusters in human, one on 5p15.33 and the other one on 16q12.2, respectively, and both the organization and the structure of the genes are highly conserved. An open reading frame coding for an unknown protein is identified in the promoter of IRXA2 on chromosome 5p. This new gene is composed of four exons and it is orientated in a head-to-head manner to IRXA2. Only a short 851 bp segment separates the two translation start codons and the two genes may share a bi-directional promoter. This bi-directional promoter is embedded in a large CpG-island, that also continues into both genes. RT-PCR analysis of the new gene reveals two alternative mRNA transcripts and a third mRNA transcript can be predicted from EST clones. The expression profile of the gene analysed in 9 different human tissues reveals that it is expressed in a coordinated fashion with IRXA2, which has led to the name CEI (Coordinated Expression to IRXA2). The CEI protein lacks homology to any known protein or protein domain in public databases, and a putative amino terminal signal peptide suggests the protein is secreted or ER compartment located. The gene is only found in the human and the chimpanzee genome, but not in the mouse or the rat genome, which suggests that CEI is unique for higher primates. As the identified bi-directional promoter not being a relic of an ancient compact genome, CEI may play an important role in the evolution of higher primates in coordination with the IRX genes.

Amino Acid Sequence↗
PubMed2001

Comparative analysis of the ETV6 gene in vertebrate genomes from pufferfish to human.

The ETV6 gene encodes an Ets-like transcription factor that is frequently rearranged in leukemias. While some of the functions of ETV6 have been uncovered recently, little is known about the key structural elements involved. Comparative genome analysis may provide novel insights into gene evolution and functions. In this study, we cloned and sequenced the homologue of ETV6 from the compact genome of the pufferfish Fugu rubripes (fETV6). The genomic structure of the fETV6 gene was investigated by sequence analysis of a contig of genomic clones. The fETV6 gene, composed of eight exons, spans about 15 kb and is 16 times smaller than its human counterpart mainly because of the reduced intron size. Three of the seven introns of fETV are unusually large (more than 2 kb), including the 8.2 kb intron 2. The gene codes for a protein of 465 amino acids that is highly related to its human homologue, exhibiting an overall identity of 58% (72% similarity). To investigate the functional and evolutionary aspects of ETV6, we undertook a comparative analysis of this gene from various vertebrates (human, mouse, chicken, zebrafish and Fugu). As expected, the PNT and ETS domains were highly conserved, with on average 81 and 95% peptide sequence identity, respectively. In addition, we found several new highly conserved regions within the central section of the protein that are likely to represent further functional or structural domains, which may be associated with the transcription repression capacity of this protein. We also found conserved putative regulatory elements in the promoter as well as in the large intron 2 of fETV6. The information derived from this comparative analysis will serve as the basis for more precise functional studies of ETV6 gene regulation and function.

Amino Acid Sequence↗
PubMed2002

Rapid screening of a novel arrayed medaka (Oryzias latipes) cosmid library.

Medaka (Oryzias latipes) has many advantages for genetic and developmental studies. With recent advances in the genome analyses of other species, rapid accumulation of resources for medaka genomics is expected. In this study, we generated an arrayed medaka cosmid library from the HNI inbred strain, carrying a 40-kb insert on average. The library consists of approximately 120,000 clones with a 6-fold genomic coverage. Cosmid clones can be screened within 2 days using standard polymerase chain reaction. Considering the advantage of the cosmid insert size and the compact genome size of the medaka, this library provides a powerful tool for future genome analyses.

Journal Article↗
PubMed1993

Electrophoretic analysis of the nuclear and organellar genomes in the ultra-small alga Cyanidioschyzon merolae.

Electrophoretic analysis reveals that the nucleus of the ultra-small eukaryotic alga Cyanidioschyzon merolae contains approximately 11.7 x 10(6) base pairs (11.7 Mb) of DNA. This compact genome is fragmented into 15 small chromosomes ranging in size from 410 to 1700 kb. The migratory behaviour of chloroplast DNA is consistent with the presence of a circular plastid genome of about 170 kb. The conformation of mitochondrial DNA resembles that in yeasts and fungi and is predominantly linear and heterogenous in size.

Cell Nucleus↗
PubMed2002

DNA loss and evolution of genome size in Drosophila.

Mutation is often said to be random. Although it must be true that mutation is ignorant about the adaptive needs of the organism and thus is random relative to them as a rule, mutation is not truly random in other respects. Nucleotide substitutions, deletions, insertions, inversions, duplications and other types of mutation occur at different rates and are effected by different mechanisms. Moreover the rates of different mutations vary from organism to organism. Differences in mutational biases, along with natural selection, could impact gene and genome evolution in important ways. For instance, several recent studies have suggested that differences in insertion/deletion biases lead to profound differences in the rate of DNA loss in animals and that this difference per se can lead to significant changes in genome size. In particular, Drosophila melanogaster appears to have a very high rate of deletions and the correspondingly high rate of DNA loss and a very compact genome. To assess the validity of these studies we must first assess the validity of the measurements of indel biases themselves. Here I demonstrate the robustness of indel bias measurements in Drosophila, by comparing indel patterns in different types of nonfunctional sequences. The indel pattern and the high rate of DNA loss appears to be shared by all known nonfunctional sequences, both euchromatic and heterochromatic, transposable and non-transposable, repetitive and unique. Unfortunately all available nonfunctional sequences are untranscribed and thus effects of transcription on indel bias cannot be assessed. I also discuss in detail why it is unlikely that natural selection for or against DNA loss significantly affects current estimates of indel biases.

Animals↗
PubMed2003

Integrated mapping, chromosomal sequencing and sequence analysis of Cryptosporidium parvum.

The apicomplexan Cryptosporidium parvum is one of the most prevalent protozoan parasites of humans. We report the physical mapping of the genome of the Iowa isolate, sequencing and analysis of chromosome 6, and approximately 0.9 Mbp of sequence sampled from the remainder of the genome. To construct a robust physical map, we devised a novel and general strategy, enabling accurate placement of clones regardless of clone artefacts. Analysis reveals a compact genome, unusually rich in membrane proteins. As in Plasmodium falciparum, the mean size of the predicted proteins is larger than that in other sequenced eukaryotes. We find several predicted proteins of interest as potential therapeutic targets, including one exhibiting similarity to the chloroquine resistance protein of Plasmodium. Coding sequence analysis argues against the conventional phylogenetic position of Cryptosporidium and supports an earlier suggestion that this genus arose from an early branching within the Apicomplexa. In agreement with this, we find no significant synteny and surprisingly little protein similarity with Plasmodium. Finally, we find two unusual and abundant repeats throughout the genome. Among sequenced genomes, one motif is abundant only in C. parvum, whereas the other is shared with (but has previously gone unnoticed in) all known genomes of the Coccidia and Haemosporida. These motifs appear to be unique in their structure, distribution and sequences.

Animals↗
PubMed2006

Histone acetylation-mediated chromatin compaction during mouse spermatogenesis.

One of the most dramatic chromatin remodelling events takes place during mammalian spermatogenesis involving massive incorporation of somatic and testis-specific histone variants, as well as generalized histone modifications before their replacement by new DNA packaging proteins. Our data suggest that the induced histone acetylation occurring after meiosis may direct the first steps of genome compaction. Indeed, a double bromodomain-containing protein expressed in postmeiotic cells, Brdt, shows the extraordinary capacity to specifically condense acetylated chromatin in vivo and in vitro. In elongating spermatids, Brdt widely co-localizes with acetylated histones before accumulating in condensed chromatin domains. These domains preferentially maintain their acetylation status until late spermatogenesis. Based on these data, we propose that Brdt mediates a general histone acetylation-induced chromatin compaction and also maintains differential acetylation of specific regions, and is therefore involved in organizing the spermatozoon's genome.

Acetylation↗
PubMed1998

A genome-based approach for the identification of essential bacterial genes.

We have used comparative genomics to identify 26 Escherichia coli open reading frames that are both of unknown function (hypothetical open reading frames or y-genes) and conserved in the compact genome of Mycoplasma genitalium. Not surprisingly, these genes are broadly conserved in the bacterial world. We used a markerless knockout strategy to screen for essential E. coli genes. To verify this phenotype, we constructed conditional mutants in genes for which no null mutants could be obtained. In total we identified six genes that are essential for E. coli (yhbZ, ygjD, ycfB, yfil, yihA, and yjeQ). The respective orthologs of the genes yhbZ, ygjD, ycfB, yjeQ, and yihA are also essential in Bacillus subtilis. This low number of essential genes was unexpected and might be due to a characteristic of the versatile genomes of E. coli and B. subtilis that is comparable to the phenomenon of nonorthologous gene displacement. The gene ygjD, encoding a sialoglycoprotease, was eliminated from a minimal genome computationally derived from a comparison of the Haemophilus influenzae and M. genitalium genomes. We show that ygjD and its ortholog ydiE are essential in E. coli and B. subtilis, respectively. Thus, we include this gene in a minimal genome. This study systematically integrates comparative genomics and targeted gene disruptions to identify broadly conserved bacterial genes of unknown function required for survival on complex media.

Amino Acid Sequence↗
PubMed1999

The plastid genome of the cryptophyte alga, Guillardia theta: complete sequence and conserved synteny groups confirm its common ancestry with red algae.

The plastid genome of the cryptophyte alga Guillardia theta (121,524 bp) has been completely sequenced. The genome is 33% G+C and contains a short, nonidentical inverted repeat (4.9 kb) encoding the two rRNA cistrons. The large and small single-copy regions are 96.3 and 15.4 kb, respectively. Forty-six genes encoding proteins for photosynthesis, 5 genes for biosynthetic function, 5 genes involved in replication and division, 30 tRNA genes, 44 ribosomal protein genes (26 large subunit and 18 small subunit), 3 translation factors, 8 genes encoding components of the transcriptional machinery including 3 ycfs (hypothetical chloroplast frames), and 26 additional ycfs have been identified. There are eight ORFs larger than 50 amino acids, 3 of which have homologues on the plastid genome of the rhodophyte, Porphyra purpurea (Reith and Munholland 1995) and/or the Synechocystis genome (Kaneko et al. 1996) and can be designated new ycfs. Intergenic spacers are very short, no introns have been detected, and several genes overlap, all resulting in a very compact genome. In addition, large clusters of genes (such as those for the ribosomal proteins) are organized into single transcriptional units (Wang et al. 1997), again resulting in an economically organized genome. The cryptophyte plastid genome is almost completely comprised of clusters of genes that are found on the rhodophyte Porphyra purpurea, confirming its common ancestry with red algae. Furthermore, recombination events involving both tRNA genes and the rRNA cistrons appear to have been responsible for the structure of the cryptophyte plastid genome, including the formation of the inverted repeat.

Cell Division↗
PubMed1996

Histones and chromatin structure in hyperthermophilic Archaea.

HMf is a histone from the hyperthermophile Methanothermus fervidus. It is the archetype and most studied member of a family of archaeal histones that have primary sequences and three-dimensional structures in common with the eukaryal nucleosome core histones and that bind and compact DNA molecules into nucleosome-like structures (NLS). HMf preparations are mixtures of two similar, small (approximately 7.5 kDa) polypeptides designated HMfA and HMfB that in vivo form both homodimers and heterodimers. HMfA synthesis predominates during exponential growth but the relative amount of HMfB increases as M. fervidus cells enter the stationary growth phase. Analyses of homogeneous preparations of recombinant (r) (HMfA)2 and (rHMfB)2 have demonstrated that these proteins have different DNA-binding and compaction properties in vitro, consistent with different roles in vivo for the (HMfA)2, (HMfB)2 and HMfA. HmfB dimers, and for the NLS that they form, in regulating gene expression and in genome compaction and stability.

Amino Acid Sequence↗
PubMed2006

Mitochondrial genome sequence evolution in Chlamydomonas.

The mitochondrial genomes of the Chlorophyta exhibit significant diversity with respect to gene content and genome compactness; however, quantitative data on the rates of nucleotide substitution in mitochondrial DNA, which might help explain the origin of this diversity, are lacking. To gain insight into the evolutionary forces responsible for mitochondrial genome diversification, we sequenced to near completion the mitochondrial genome of the chlorophyte Chlamydomonas incerta, estimated the evolutionary divergence between Chlamydomonas reinhardtii and C. incerta mitochondrial protein-coding genes and rRNA-coding regions, and compared the relative evolutionary rates in mitochondrial and nuclear genes. Synonymous and nonsynonymous substitution rates do not differ significantly between the mitochondrial and nuclear protein-coding genes. The mitochondrial rRNA-coding regions, however, are evolving much faster than their nuclear counterparts, and this difference might be explained by relaxed functional constraints on the mitochondrial translational apparatus due to the small number of proteins synthesized in Chlamydomonas mitochondria. Substitution rates at synonymous sites in a nonstandard mitochondrial gene (rtl) and at intronic and synonymous sites in nuclear genes expressed at low levels suggest that the mutation rate is similar in these two genetic compartments. Potential evolutionary forces shaping mitochondrial genome evolution in Chlamydomonas are discussed.

Animals↗
PubMed2004

Genome-wide comparative analysis of the transposable elements in the related species Arabidopsis thaliana and Brassica oleracea.

Transposable elements (TEs) are the major component of plant genomes where they contribute significantly to the >1,000-fold genome size variation. To understand the dynamics of TE-mediated genome expansion, we have undertaken a comparative analysis of the TEs in two related organisms: the weed Arabidopsis thaliana (125 megabases) and Brassica oleracea ( approximately 600 megabases), a species with many crop plants. Comparison of the whole genome sequence of A. thaliana with a partial draft of B. oleracea has permitted an estimation of the patterns of TE amplification, diversification, and loss that has occurred in related species since their divergence from a common ancestor. Although we find that nearly all TE lineages are shared, the number of elements in each lineage is almost always greater in B. oleracea. Class 1 (retro) elements are the most abundant TE class in both species with LTR and non-LTR elements comprising the largest fraction of each genome. However, several families of class 2 (DNA) elements have amplified to very high copy number in B. oleracea where they have contributed significantly to genome expansion. Taken together, the results of this analysis indicate that amplification of both class 1 and class 2 TEs is responsible, in part, for B. oleracea genome expansion since divergence from a common ancestor with A. thaliana. In addition, the observation that B. oleracea and A. thaliana share virtually all TE lineages makes it unlikely that wholesale removal of TEs is responsible for the compact genome of A. thaliana.

Arabidopsis↗
PubMed2002

Molecular biology of Borna disease virus and persistence.

Borna disease virus (BDV) causes central nervous system (CNS) disease that is frequently manifested by behavioral abnormalities. Recent evidence indicates that the natural host range and geographic distribution of BDV is wider than originally thought BDV has been molecularly characterized as a non-segmented, negative single-stranded (NNS) RNA virus. Its genome (ca 8.9 kb), the smallest among known NNS RNA viruses, has an organization similar to that of other members of the order Mononegavirales. BDV has the property, unique among known animal NNS RNA viruses, of a nuclear site for the replication and transcription of its genome. The nucleocytoplasmic transport of BDV macromolecules is an essential component of the life cycle of BDV. An overlap of transcription units and transcriptive signals, overlap of ORFs, transcriptional readthrough and RNA splicing regulate expression of the BDV compact genome. The concurrent use of such diversity of strategies for the regulation of virus gene expression is unique among known NNS RNA viruses. Moreover, BDV appears to have also an unusual assembly process. Based on its unique genetic and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. Therefore, the investigation of the molecular biology of BDV may provide new insights about the biology of mononegaviruses, which include important human pathogens.

Animals↗
PubMed1988

Genome organization of the killer plasmid pGK12 from Kluyveromyces lactis.

We have determined the entire sequence of the plasmid K2 from Kluyveromyces lactis which is involved in the maintenance of both killer plasmids in the cell. K2 shares many of the characteristics of the smaller killer plasmid K1: high A+T content (74.7%) and very compact genomic organization. K2 contains ten open reading frames. Some of them overlap on different strands and some on the same strand. Northern blotting of K2 transcripts shows that at least eight ORFs are transcribed. Analysis of the predicted aminoacid sequence of ORF2 from K2 reveals homology with the aminoacid sequence of ORF 1 from K1 and with several viral DNA polymerases. The sequence of K2 from Saccaromyces cerevisiae F102-2 was also determined. Only one nucleotide difference was found between the K2 sequence from the two yeasts. This mutation does not change the genome organization of the plasmid and has only minimal effect on the structure of the encoded proteins.

Amino Acid Sequence↗