Yun Liu

The formation of tetraploid stocks of red crucian carp×common carp hybrids as an effect of interspecific hybridization

Abstract F 3 –F 8 hybrids of red crucian carp ( Carassius auratus red var.) (♀)×common carp ( Cyprinus carpio L.) (♂) were found to be allotetraploids by examining the numbers and karyotypes of chromosomes, the DNA content of red blood cells, and the mean nuclear erythrocyte volumes. The chromosome numbers of F 3 –F 8 were 4 n =200. The chromosome numbers of two types of triploids produced by mating F 3 –F 8 (♂), respectively, with diploid Japanese crucian carp ( C. auratus cuvieri T. et S.) (♀) and Xingguo red carp (♀) were 3 n =150. The mean DNA content of F 3 and the triploid are, respectively, two times and 1.5 times of that of diploid Japanese crucian carp. F 3 hybrids had mean erythrocyte nuclear volumes about two times larger than diploid Japanese crucian carp. The hybrids F 3 –F 8 were fertile. It is easy for F 3 –F 8 to produce their tetraploid offspring. The triploids were sterile. Based on the analysis of karyotype and the intermediate appearance with two pairs of small bases of barbels, F 3 –F 8 hybrids were proved to be allotetraploids with two chromosome sets of red crucian carp and two chromosome sets of common carp. It is concluded that the females and males of F 2 hybrids were able to produce, respectively, diploid eggs and diploid spermatozoa and they fertilized to form tetraploid fish.

Comparative analysis of the mitochondrial DNA control region in cyprinids with different ploidy level

The comprehensive analysis of the complete mitochondrial DNA control region (mtDNA-CR) sequences from the different ploidy level cyprinid fish was made. The mtDNA-CR sequences for allotriploid crucian carp and allotriploid common carp were amplified by the polymerase chain reaction (PCR) and the amplified products were sequenced. The mtDNA-CR sequences from diploids, such as the red crucian carp, common carp, Japanese crucian carp, and zebrafish to polyploids, such as the allotetraploid, allotriploid crucian carp, and allotriploid common carp, were compared and analyzed. The extended terminal associated sequences (ETAS), central conserved sequence blocks (CSB-F, CSB-E, CSB-D), conserved sequence blocks (CSB-1, CSB-2, CSB-3), and putative promoters of the mtDNA-CR in the seven cyprinid fish were observed. Our data were consistent with the opinion of three domains (extended terminal associated sequences, central, conserved sequence block domains) within the mtDNA-CR in the mammals. The most conserved region in the mtDNA-CR was the central conserved sequence block domain (34.06% of nucleotide positions variable), whereas in the ETAS and CSB domains, 66.91% and 40.00% of nucleotide positions were variable, respectively. Based on the comparative analysis of the mtDNA-CR in cyprinids with different ploidy level, the consensus sequences of functional conserved units of the mtDNA-CR in cyprinids were identified. These results provided the useful and important information for the further study of the mtDNA-CR structure in fish. The similarities of the mtDNA-CR sequences between the allotriploid crucian carp and allotriploid common carp and their female parents were higher than those between allotriploid crucian carp and allotriploid common carp and their male parents, providing the direct evidence of the maternal inheritance of the mtDNA-CR in the allotriploid crucian carp and allotriploid common carp.

The formation of the polyploid hybrids from different subfamily fish crossings and its evolutionary significance.

This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp × blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (♀) × blunt snout bream (BSB) (♂) as well as their pentaploid hybrids. The triploid hybrids possessed 124 chromosomes with two sets from RCC and one set from BSB; the tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from BSB. The females of tetraploid hybrids produced unreduced tetraploid eggs that were fertilized with the haploid sperm of BSB to generate pentaploid hybrids with 172 chromosomes with three sets from BSB and two sets from RCC. The ploidy levels of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers. In addition, this study also presents results about the phenotypes and feeding habits of polyploid hybrids and discusses the formation mechanism of the polyploid hybrids. It is the first report on the formation of the triploid, tetraploid, and pentaploid hybrids by crossing parents with a different chromosome number in vertebrates. The formation of the polyploid hybrids is potentially interesting in both evolution and fish genetic breeding.

Coexistence of diploid, triploid and tetraploid crucian carp (Carassius auratus) in natural waters

BackgroundCrucian carp (abbreviated CC) belongs to the genus of Carassius within the family of Cyprinidae. It has been one of the most important freshwater species for Chinese aquaculture and is especially abundant in the Dongting water system of Hunan province. CC used to be considered as all diploid forms. However, coexistence of diploid (abbreviated 2nCC), triploid (abbreviated 3nCC) and tetraploid crucian carp (abbreviated 4nCC) population of the Dongting water system was first found by our recently researches.ResultsWe examined the ploidy level and compared biological characteristics in different ploidy CC. In reproductive mode, 2nCC was bisexual generative and 4nCC generated all-female offspring by gynogenesis. However, 3nCC generated progenies in two different ways. 3nCC produced bisexual triploid offspring fertilized with 3nCC spermatozoa, while it produced all-female triploid offspring by gynogenesis when its ova were activated by heterogenous spermatozoa. The complete mitochondrial DNA of three different ploidy fishes was sequenced and analyzed, suggesting no significant differences. Interestingly, microchromosomes were found only in 3nCC, which were concluded to be the result of hybridization. Allogenetic DNA fragments of Sox genes were obtained in 3nCC and 4nCC, which were absent in 2nCC. Phylogenetics analysis based on Sox4 gene indicated 3nCC and 4nCC formed a separate group from 2nCC.ConclusionsIn summary, this is the first report of the co-existence of three types of different ploidy crucian carps in natural waters in China. It was proved that the coexistence of different ploidy CC was reproductively maintained. We further hypothesized that 3nCC and 4nCC were allopolyploids that resulted from hybridization. The different ploidy CC population we obtained in this study possesses great significance for the study of polyploidization and the evolution of vertebrates.

Human Bcl-2 activates ERK signaling pathway to regulate activating protein-1, lens epithelium-derived growth factor and downstream genes

The proto-oncogene, bcl-2, has various functions besides its role in protecting cells from apoptosis. One of the functions is to regulate expression of other genes. Previous studies have demonstrated that Bcl-2 regulates activities of several important transcription factors including NF-κB and p53, and also their downstream genes. In our recent studies, we reported that Bcl-2 substantially downregulates expression of the endogenous αB-crystallin gene through modulating the transcriptional activity of lens epithelium-derived growth factor (LEDGF). In the present communication, we report that human Bcl-2 can positively regulate expression of the proto-oncogenes c-jun and c-fos. Moreover, it enhances the DNA binding activity and transactivity of the activating protein-1 (AP-1). Furthermore, we present evidence to show that Bcl-2 can also activate both ERK1 and ERK2 MAP kinases. Inhibition of the activities of these kinases or the upstream activating kinases by pharmacological inhibitors or dominant-negative mutants abolishes the Bcl-2-mediated regulation of AP-1, LEDGF and their downstream genes. Together, our results demonstrate that through activation of the ERK kinase signaling pathway, Bcl-2 regulates the transcriptional activities of multiple transcription factors, and hence modulates the expression of their downstream genes. Thus, our results provide a mechanism to explain how Bcl-2 may regulate expression of other genes.

Analysis of 5S rDNA organization and variation in polyploid hybrids from crosses of different fish subfamilies

In this article, sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) were conducted in red crucian carp (RCC), blunt snout bream (BSB), and their polyploid offspring. Three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp) of RCC were characterized by distinct NTS types (designated NTS-I, II, and III for the 83, 220, and 357 bp monomers, respectively). In BSB, only one monomeric 5S rDNA was observed (designated class IV: 188 bp), which was characterized by one NTS type (designated NTS-IV: 68 bp). In the polyploid offspring, the tetraploid (4nRB) hybrids partially inherited 5S rDNA classes from their female parent (RCC); however, they also possessed a unique 5S rDNA sequence (designated class I-L: 203 bp) with a novel NTS sequence (designated NTS-I-L: 83 bp). The characteristic paternal 5S rDNA sequences (class IV) were not observed. The 5S rDNA of triploid (3nRB) hybrids was completely inherited from the parental species, and generally preserved the parental 5S rDNA structural organization. These results first revealed the influence of polyploidy on the organization and evolution of the multigene family of 5S rDNA of fish, and are also useful in clarifying aspects of vertebrate genome evolution.

Protein Phosphatase-1 Modulates the Function of Pax-6, a Transcription Factor Controlling Brain and Eye Development

Pax-6 is an evolutionarily conserved transcription factor and acts high up in the regulatory hierarchy controlling eye and brain development in humans, mice, zebrafish, and Drosophila. Previous studies have shown that Pax-6 is a phosphoprotein, and its phosphorylation by ERK, p38, and homeodomain-interacting protein kinase 2 greatly enhances its transactivation activity. However, the protein phosphatases responsible for the dephosphorylation of Pax-6 remain unknown. Here, we present both in vitro and in vivo evidence to show that protein serine/threonine phosphatase-1 is a major phosphatase that directly dephosphorylates Pax-6. First, purified protein phosphatase-1 directly dephosphorylates Pax-6 in vitro. Second, immunoprecipitation-linked Western blot revealed that both protein phosphatase-1α and protein phosphatase-1β interact with Pax-6. Third, overexpression of protein phosphatase-1 in human lens epithelial cells leads to dephosphorylation of Pax-6. Finally, inhibition of protein phosphatase-1 activity by calyculin A or knockdown of protein phosphatase-1α and protein phosphatase-1β by RNA interference leads to enhanced phosphorylation of Pax-6. Moreover, our results also demonstrate that dephosphorylation of Pax-6 by protein phosphatase-1 significantly modulates its function in regulating expression of both exogenous and endogenous genes. These results demonstrate that protein phosphatase 1 acts as a major phosphatase to dephosphorylate Pax-6 and modulate its function.

Human Telomerase Reverse Transcriptase Immortalizes Bovine Lens Epithelial Cells and Suppresses Differentiation through Regulation of the ERK Signaling Pathway

Telomerase is a specialized reverse transcriptase that extends telomeres of eukaryotic chromosomes. The functional telomerase complex contains a telomerase reverse transcriptase catalytic subunit and a telomerase template RNA. We have previously demonstrated that human telomerase reverse transcriptase (hTERT) catalytic subunit is functionally compatible with a telomerase template RNA from rabbit. In this study, we show that hTERT is also functionally compatible with a telomerase template RNA from bovine. Introduction of hTERT into bovine lens epithelial cells (BLECs) provides the transfected cells telomerase activity. The expressed hTERT in BLECs supports normal growth of the transfected cells for 108 population doublings so far, and these cells are still extremely healthy in both morphology and growth. In contrast, the vector-transfected cells display growth crisis after 20 population doublings. These cells run into cellular senescence due to shortening of the telomeres and also commit differentiation as indicated by the accumulation of the differentiation markers, β-crystallin and filensin. hTERT prevents the occurrence of both events. By synthesizing new telomere, hTERT prevents replicative senescence, and through regulation of MEK/ERK, protein kinase C, and protein kinase A and eventual suppression of the MEK/ERK signaling pathway, hTERT inhibits differentiation of BLECs. Our finding that hTERT can suppress RAS/RAF/MEK/ERK signaling pathway to prevent differentiation provides a novel mechanism to explain how hTERT regulates cell differentiation.

Organization and Variation Analysis of 5S rDNA in Different Ploidy-level Hybrids of Red Crucian Carp × Topmouth Culter

Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (Carassius auratus red var., RCC♀, Cyprininae, 2n = 100) × topmouth culter (Erythroculter ilishaeformis Bleeker, TC♂, Cultrinae, 2n = 48) were successfully produced. Diploid hybrids possessed 74 chromosomes with one set from RCC and one set from TC; triploid hybrids harbored 124 chromosomes with two sets from RCC and one set from TC; tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from TC. The 5S rDNA of the three different ploidy-level hybrids and their parents were sequenced and analyzed. There were three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp) in RCC and two monomeric 5S rDNA classes (designated class IV: 188 bp, and class V: 286 bp) in TC. In the hybrid offspring, diploid hybrids inherited three 5S rDNA classes from their female parent (RCC) and only class IV from their male parent (TC). Triploid hybrids inherited class II and class III from their female parent (RCC) and class IV from their male parent (TC). Tetraploid hybrids gained class II and class III from their female parent (RCC), and generated a new 5S rDNA sequence (designated class I–N). The specific paternal 5S rDNA sequence of class V was not found in the hybrid offspring. Sequence analysis of 5S rDNA revealed the influence of hybridization and polyploidization on the organization and variation of 5S rDNA in fish. This is the first report on the coexistence in vertebrates of viable diploid, triploid and tetraploid hybrids produced by crossing parents with different chromosome numbers, and these new hybrids are novel specimens for studying the genomic variation in the first generation of interspecific hybrids, which has significance for evolution and fish genetics.

Elevated expressions of GH/IGF axis genes in triploid crucian carp.

Growth hormone (GH), growth hormone receptor (GHR) and insulin-like growth factor 1 (IGF-1) are pivotal signaling factors of the GH/IGF axis, which plays a crucial role in regulating growth in vertebrates. In this study, GH, GHR and IGF-1 cDNAs were cloned from triploid and tetraploid crucian carp. In addition, mRNA expression levels were characterized in diploid red crucian carp, triploids and tetraploids. Reverse transcriptase PCR indicated that GH genes were only expressed in the pituitary, while GHR and IGF-1 were widely expressed in all tested tissues. Real-time PCR study of different seasonal profiles showed that triploids had significantly higher expression of the studied genes during both the prespawning and the spawning season. Although different temperatures (22, 26 and 30°C) showed no significant effects on GH, GHR and IGF-1 mRNA expression in either diploids or triploids, triploids had higher expression levels than diploids at each temperature. After 1 week of fasting, the expression of all studied genes was reduced in both diploids and triploids, while the expressions levels were higher in triploids than in diploids. These results suggest that the elevated expression of GH/IGF axis genes in triploids plays a crucial role in the faster growth rate of triploids.