A de novo originated gene depresses budding yeast mating pathway and is repressed by the protein encoded by its antisense strand

Recent transcription profiling studies have revealed an unexpectedly large proportion of antisense transcripts in eukaryotic genomes. These antisense genes seem to regulate gene expression by interacting with sense genes. Previous studies have focused on the non-coding antisense genes, but the possible regulatory role of the antisense protein is poorly understood. In this study, we found that a protein encoded by the antisense gene ADF1 acts as a transcription suppressor, regulating the expression of sense gene MDF1 in Saccharomyces cerevisiae. Based on the evolutionary, genetic, cytological and biochemical evidence, we show that the protein-coding sense gene MDF1 most likely originated de novo from a previously non-coding sequence and can significantly suppress the mating efficiency of baker's yeast in rich medium by binding MAT alpha 2 and thus promote vegetative growth. These results shed new light on several important issues, including a new sense-antisense interaction mechanism, the de novo origination of a functional gene, and the regulation of yeast mating pathway.

The sequence and de novo assembly of the giant panda genome

Using next-generation sequencing technology alone, we have successfully generated and assembled a draft sequence of the giant panda genome. The assembled contigs (2.25 gigabases (Gb)) cover approximately 94% of the whole genome, and the remaining gaps (0.05 Gb) seem to contain carnivore-specific repeats and tandem repeats. Comparisons with the dog and human showed that the panda genome has a lower divergence rate. The assessment of panda genes potentially underlying some of its unique traits indicated that its bamboo diet might be more dependent on its gut microbiome than its own genetic composition. We also identified more than 2.7 million heterozygous single nucleotide polymorphisms in the diploid genome. Our data and analyses provide a foundation for promoting mammalian genetic research, and demonstrate the feasibility for using next-generation sequencing technologies for accurate, cost-effective and rapid de novo assembly of large eukaryotic genomes.

New insights into the karyotypic relationships of Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis)

To investigate the karyotypic relationships between Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis), a complete set of Chinese muntjac chromosome-specific painting probes has been assigned to G-banded chromosomes of these three species. Sixteen autosomal probes (i.e. 6-10, 12-22) of the Chinese muntjac each delineated one pair of conserved segments in the forest musk deer and gayal, respectively. The remaining six autosomal probes (1-5, and 11) each delineated two to five pairs of conserved segments. In total, the 22 autosomal painting probes of Chinese muntjac delineated 33 and 34 conserved chromosomal segments in the genomes of forest musk deer and gayal, respectively. The combined analysis of comparative chromosome painting and G-band comparison reveals that most interspecific homologous segments show a high degree of conservation in G-banding patterns. Eleven chromosome fissions and five chromosome fusions differentiate the karyotypes of Chinese muntjac and forest musk deer; twelve chromosome fissions and six fusions are required to convert the Chinese muntjac karyotype to that of gayal; one chromosome fission and one fusion separate the forest musk deer and gayal. The musk deer has retained a highly conserved karyotype that closely resembles the proposed ancestral pecoran karyotype but shares none of the rearrangements characteristic for the Cervidae and Bovidae. Our results substantiate that chromosomes 1-5 and 11 of Chinese muntjac originated through exclusive centromere-to-telomere fusions of ancestral acrocentric chromosomes. Copyright (C) 2005 S. Karger AG, Basel.

Fish-Specific Duplicated dmrt2b Contributes to a Divergent Function through Hedgehog Pathway and Maintains Left-Right Asymmetry Establishment Function

Gene duplication is thought to provide raw material for functional divergence and innovation. Fish-specific dmrt2b has been identified as a duplicated gene of the dmrt2a/terra in fish genomes, but its function has remained unclear. Here we reveal that Dmrt2b knockdown zebrafish embryos display a downward tail curvature and have U-shaped somites. Then, we demonstrate that Dmrt2b contributes to a divergent function in somitogenesis through Hedgehog pathway, because Dmrt2b knockdown reduces target gene expression of Hedgehog signaling, and also impairs slow muscle development and neural tube patterning through Hedgehog signaling. Moreover, the Dmrt2b morphants display defects in heart and visceral organ asymmetry, and, some lateral-plate mesoderm (LPM) markers expressed in left side are randomized. Together, these data indicate that fish-specific duplicated dmrt2b contributes to a divergent function in somitogenesis through Hedgehog pathway and maintains the common function for left-right asymmetry establishment.

Origin and evolution of the RIG-I like RNA helicase gene family

Background: The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. Results: The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. Conclusion: The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

Characterization of complete genome sequence of the spring viremia of carp virus isolated from common carp (Cyprinus carpio) in China

The complete genome of spring viraemia of carp virus (SVCV) strain A-1 isolated from cultured common carp (Cyprinus carpio) in China was sequenced and characterized. Reverse transcription-polymerase chain reaction (RT-PCR) derived clones were constructed and the DNA was sequenced. It showed that the entire genome of SVCV A-1 consists of 11,100 nucleotide base pairs, the predicted size of the viral RNA of rhabdoviruses. However, the additional insertions in bp 4633-4676 and bp 4684-4724 of SVCV A-1 were different from the other two published SVCV complete genomes. Five open reading frames (ORFs) of SVCV A-1 were identified and further confirmed by RT-PCR and DNA sequencing of their respective RT-PCR products. The 5 structural proteins encoded by the viral RNA were ordered 3'-N-P-M-G-L-5'. This is the first report of a complete genome sequence of SVCV isolated from cultured carp in China. Phylogenetic analysis indicates that SVCV A-1 is closely related to the members of the genus Vesiculovirus, family Rhabdoviridae.

Identification of novel SINEs from Cyprinidae and their evolutionary significance

Short interspersed nuclear elements (SINEs) are widespread among eukaryotic genomes. They are repetitive DNA sequences that have been amplified by retrotransposition. In this study, a class of SINEs were isolated from the Opsariichthys bidens genome, and named Opsar. Sequence analysis confirmed that Opsar is a new class of typical SINEs derived from tRNA molecules. With the tRNA-derived region of Opsar and through BLASTN search, we further identified Zb-SINEs from the zebrafish genome, which includes two groups: Zb-SINE-A and Zb-SINE-B. The Zb-SINE-A group comprises subfamilies of -Al--A5, and the Zb-SINE-B group is a dimer of the tRNA(Ala)-derived region and shares a similar dimeric composition to Alu. Zb-SINEs are composed of three distinct regions: a 5 end tRNA-derived region, a tRNA-unrelated region and a 3 end AT-rich region. The flanking regions are AT rich. The average length of Zb-SINEs elements is about 340 6p. Zb-SINEs account for as much as 0.1% of the whole zebrafish genome. About 70% of the Zb-SINEs are on chromosomes 11, 18, and 19. These Zb-SINEs were characterized by PCR and dot hybridization. The distribution pattern of Zb-SINEs in genome strongly supports the master genes model. The tRNA-derived regions of Opsar and Zb-SINEs were compared with the tRNA(Ala) gene, and they showed 76% similarity, indicating that Opsar and Zb-SINEs originated from an inactive tRNA(Ala) sequence or a tRNA(Ala)-like sequence. In view of the evolutionary status of zebrafish in the Cyprinidae, we deduced that Zb-SINEs were a very old class of interspersed sequences.

Molecular characterization of three Rana grylio virus (RGV) isolates and Paralichthys olivaceus lymphocystis disease virus (LCDV-C) in iridoviruses

Three Rana grylio virus (RGV) isolates and lymphocystis disease virus (LCDV-C) were molecularly characterized by antigenicity comparison, Western blot detection of viral polypeptides, restriction fragment length polymorphism analysis of viral genomes, and MCP sequence analysis. Significant antigenicity differences existed among the three RGV isolates and LCDV-C. Western blot detection indicated that the viral polypeptides of three RGV isolates could be recognized by the anti-RGV9807 serum, whereas no bands were observed in the LCDV-C, and significant differences exist among the band patterns of three RGV isolates. Restriction fragment length polymorphism (RFLP) analysis was performed by digesting genomic DNA of the four iridovirus isolates with restriction endonucleases HindIII, KpnI, XbaI and BamHI. On the whole, obvious discrepancies existed between LCDV-C and RGV isolates, and some significant band pattern differences were also revealed between RGV9808 and RGV9506 (or RGV9807) in the profiles of restriction endonucleases Xbal, Kpn I and BamHI. PCR amplification and sequence analysis of MCP gene sequence further revealed their phylogenetic relationship among the three RGV isolates, LCDV-C and other iridoviruses. RGV9506, RGV9807 and RGV9808 are clustered together with other ranaviruses, such as FV3, BIV, TFV and ENHV, although the RGV9808 is more close to EHNV than to other ranaviruses. Additionally, LCDV-C is clustered with LCDV-1, the type species of genus Lymphocystisvirus. The current study provides clear evidence that significant genetic difference exists among the three RGV isolates. Therefore, further work on comparative genomic studies will contribute significantly to understanding of their taxonomic position and pathological mechanism. (C) 2005 Elsevier B.V. All rights reserved.

Identification, cloning, and characterization of microsatellite DNA in Euglena gracilis

Microsatellite DNA has been developed into one of the most popular genetic markers. We have identified and cloned microsatellite loci in the genome of a free-living protozoan Euglena gracilis FACHB-848, using the random amplified microsatellites method (RAMS). The digoxigenin-labelled oligonucleotides(CT)(10) and (GT)(10) served as probes to detect complementary sequences in the randomly amplified polymorphic DNA (RAPD) fingerprints produced by means of Southern blotting. Subsequently, positive RAPD fragments were cloned. From a total of 31 RAPD primer profiles, eight microsatellite loci of E. gracilis were detected and characterized. Further, six sites (i.e. EGMS1, EGMS3, EGMS4, EGMS5, EGMS6, and EGMS7) showed polymorphisms. We found a GT or CT microsatellite every 10.5 kb in the genome of E. gracilis, and similar to animal genomes, the (GT)(n) motif was much more abundant than the (CT)(n) motif. These polymorphic microsatellite DNA will serve as advantageous molecular markers for studying the genetic diversity and molecular ecology of Euglena.

Characterization of a highly conserved microsatellite marker with utility potentials in cyprinid fishes

A microsatellite locus, MFW1, originating from common carp is highly conserved in flanking nucleotides but variable in repeat length in some fishes from different families of the Cypriniformes. This orthologous locus is polymorphic in approximately 58% of the species tested in the order and is inherited by Mendelian law. It proved to be a potentially good marker in population genetics and in the cyprinid species-breeding programme in which no microsatellite markers were available.

Cytoplasmic impact on cross-genus cloned fish derived from transgenic common carp (Cyprinus carpio) nuclei and goldfish (Carassius auratus) enucleated eggs

In previous studies of nuclear transplantation, most cloned animals were obtained by intraspecies nuclear transfer and are phenotypically identical to their nuclear donors; furthermore, there was no further report on successful fish cloning since the report of cloned zebrafish. Here we report the production of seven cross-genus cloned fish by transferring nuclei from transgenic common carp into enucleated eggs of goldfish. Nuclear genomes of the cloned fish were exclusively derived from the nuclear donor species, common carp, whereas the mitochondrial DNA from the donor carp gradually disappeared during the development of nuclear transfer (NT) embryos. The somite development process and somite number of nuclear transplants were consistent with the recipient species, goldfish, rather than the nuclear donor species, common carp. This resulted in a long-lasting effect on the vertebral numbers of the cloned fish, which belonged to the range of goldfish. These demonstrate that fish egg cytoplasm not only can support the development driven by transplanted nuclei from a distantly related species at the genus scale but also can modulate development of the nuclear transplants.

Complete genome sequence of lymphocystis disease virus isolated from China

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.

Two unisexual artificial polyploid clones constructed by genome addition of common carp (Cyprinus carp) and crucian carp (Carassius auratus)

A polyploid hybrid fish with natural gynogenesis can prevent segregation and maintain their hybrid vigor in their progenies. Supposing the reproduction mode of induced polyploid fish being natural gynogenesis, allopolyploid hybrid between common carp and crucian carp into allopolyploid was performed. The purpose of this paper is to describe a lineage from sexual diploid carp transforming into allotriploid and allotetraploid unisexual clones by genome addition. The diploid hybrid between common carp and crucian carp reproduces an unreduced nucleus consisting of two parental genomes. This unreduced female pronucleus will fuse with male pronucleus and form allotriploid zygote after penetration of related species sperms. Allotriploid embryos grow normally, and part of female allotriploid can produce unreduced mature ova with three genomes. Mature ova of most allotriploid females are provided with natural gynogenetic trait and their nuclei do not fuse with any entrance sperm. All female offspring are produced by gynogenesis of allotriploid egg under activation of penetrating sperms. These offspring maintain morphological traits of their allotriploid maternal and form an allotetraploid unisexual clone by gynogenetic reproduction mode. However, female nuclei of rare allotriploid female can fuse with penetrating male pronuclei and result in the appearance of allotetraploid individuals by means of genome addition. All allotetraploid females can reproduce unreduced mature eggs containing four genomes. Therefore, mature eggs of allotetraploid maintain gynogenetic trait and allotetraploid unisexual clone is produced under activation of related species sperms.

Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus (genus Aquareovirus, family Reoviridae)

Full-length and partial genome sequences of four members of the genus Aquareovirus, family Reoviridae (Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus) were characterized. Based on sequence comparison, the unclassified Grass carp reovirus was shown to be a member of the species Aquareovirus C The status of golden ide reovirus, another unclassified aquareovirus, was also examined. Sequence analysis showed that it did not belong to the species Aquareovirus A or C, but assessment of its relationship to the species Aquareovirus B, D, E and F was hampered by the absence of genetic data from these species. In agreement with previous reports of ultrastructural resemblance between aquareoviruses and orthoreoviruses, genetic analysis revealed homology in the genes of the two groups. This homology concerned eight of the 11 segments of the aquareovirus genome (amino acid identity 17-42%), and similar genetic organization was observed in two other segments. The conserved terminal sequences in the genomes of members of the two groups were also similar. These data are undoubtedly an indication of the common evolutionary origin of these viruses. This clear genetic relatedness between members of distinct genera is unique within the family Reoviridae. Such a genetic relationship is usually observed between members of a single genus. However, the current taxonomic classification of aquareoviruses and orthoreoviruses in two different genera is supported by a number of characteristics, including their distinct G+C contents, unequal numbers of genome segments, absence of an antigenic relationship, different cytopathic effects and specific econiches.

酵母基因组进化中一些重要新性状的产生及其效应

本论文用生物信息学的方法对酵母基因组进化中产生的新性状进行了系统 深入的研究。首先，在大多数的真核生物中，线粒体是生物能量生成所必需的细 胞器。但当葡萄糖的含量丰富的时候，即使是在有氧条件下，经过基因组重复 （WGD，whole genome duplication）后的大多酵母也都可以不需要线粒体而执行 发酵过程，而且甚至在线粒体基因组缺陷的情况下仍可以生存。在本次研究中， 我们揭示核编码的线粒体相关基因的进化速率在基因组重复后的物种中比其在 基因组重复前的物种中显著加快。而且这些基因的密码子使用偏好也在基因组重 复后的物种中减弱。密码子使用偏好的模式和一个特殊转录调控因子的分布显示 在基因组重复后的进化支系中，有效的有氧发酵过程的起源时间大致是在 Kluyveromyces polysporus 和 Saccharomyces castellii 从它们的共同祖先分化之 后。根据上述结果我们得出结论，可能正是这种新的能量策略的产生导致了线粒 体相关基因的功能在基因组重复后的物种中选择性放松。 其次，我们系统地研究了一个多细胞真菌Ashbya gossypii 和九个单细胞酵母 之间密码子使用偏好性的差异。细胞周期调控基因一直被认为是它们形态差异的 关键基因。由于A. gossypii 和典型的单细胞酵母Saccharomyces cerevisiae 有几乎 完全一样的细胞周期调控基因，因此形态上的差异可能是由于直系同源基因的表 达调控差异造成的。我们发现在A. gossypii 中细胞周期基因的翻译效率比在其他 单细胞酵母中显著增高，同时也发现单细胞酵母中的新陈代谢基因比其在A. gossypii 中有显著增高的翻译效率。因为基因的翻译效率和该基因在物种中的重 要性密切相关，所以我们观察到的这些基因翻译效率的显著差异可能可以阐明 A. gossypii 和单细胞酵母的形态差异的原因。同时我们的结果对理解真核生物多 细胞的起源过程也有提示意义。