[Microsatellite marker analysis of genetic variation between the allotetraploid crucian-carp and their original parents].

Twelve individuals from the allotetraploid crucian-carp,common carp, and red crucian carp were analyzed by using 32 pairs microsatellite primers of the poly (CA) type which were isolated from common carp (Cyprinus carpio L.) by Crooijmans et al. Electrophoretogram patterns showed that 15 pairs of microsatellite primers reproducibly produced the well-identifiable bands. The number of alleles per marker varied from 1 to 8, and the size of alleles ranged from 100 bp to 420 bp. The genetic similarity index between the allotetraploid crucian-carp and their original maternal red crucian carp was 0.5625, and the genetic similarity index between the allotetraploid crucian-carp and their original paternal common carp was 0.5125. The similarity index between red crucian carp and common carp was 0.4204. Both microsatellite and RAPD showed the similar results of the genetic similarity index. However, greater genetic distances were found among microsatellite markers than those among RAPD. These results demonstrate that the microsatellite-based analysis of the genetic variation is useful in measuring intrabiotype differences.

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, beta-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.

[Studies on the ploymorphic of sperm of F2 hybrids of red crucian carp x common carp].

AThe ultrastructures of the sperm of F2 hybrids of red crucian carp x common carp were studied by using scanning and transmission electron microscope. The sperm of the F2 hybrids consisted of head, mid-piece and tail. There was no acrosome at the anterior end of the nuclears, whereas there was a vesicle. The results revealed that there existed obviously ploymorphic in the sperm of F2 hybrids. In the water-like semen from males of F2 hybrids, different sizes of the head of the sperm including haploid, diploid, tetraploid, and aneuploid sperm were observed. The head diameter of the smallest sperm was only 1.32 microm, but that of the biggest one was about 18.39 microm, and most of them varied from 1.85 to 2.15 microm. The haploid sperm was normal, while the a-neuploid, diploid, tetraploid and multiploid sperm were abnormal. Among the abnormal sperm, there was a super sperm with about 20 tails, whose head volume was much bigger than that of any other sperm. From the results of the transmission electron microscope, 3 sperm with two nucleus and 1 sperm with two tails were found. This study provided an useful evidence for the mechanism that the formation of tetraploid in F3 hybrids was due to the fertilization of the diploid eggs and diploid sperm produced by F2 hybrids.

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-kappaB and p53, and also their downstream genes. In our recent studies, we reported that Bcl-2 substantially downregulates expression of the endogenous alphaB-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.

[Studies of F1 of transgenic allotetraploid hybrids of Carassius auratus red var. (Female) x Cyprinus carpio (Male)].

The tetraploid fish has been developed by assortative breeding the hybrids of Carassius auratus red var. (Female) x cyprinus carpio (Male), which has the stable genetic characters and can reproduce themselves. An "all-fish" recombinant DNA construct (pCAgcGHc) containing common carp beta-actin gene promoter and cDNA for grass carp (Ctenopharyngodon idella) growth hormone gene was introduced into fertilized eggs of the allotetroploid fish through microinjection as soon as artificial insemination was done. Artificial insemination was carried out between the female and the male transgenic allotetraploid fish which contain the "all-fish" recombinant DNA construct (pCAgcGHc) and are the biggest in the size. Fifty F1 samples of transgenic allotetraploid fish of 150 days and 50 allotetraploid fish (regarded as the control) were chosen, and the weight and the body length of each were measured, the results showed that F1 of transgenic allotetraploid fish of 150 days had obvious growth dominance compared with the control. Genomic DNA of tail fin was extracted from 20 F1 of transgenic allotetraploid fish of 150 days and the control. Proper primers were introduced to check whether the sample had the transgene. Pa, the upstream primer, is located in beta-actin promoter, and Pg, the downstream primer, is located in growth hormone cDNA for grass carp (gcGHc). The transgene was detected in 90% F1 of transgenic allotetraploid fish in tail fin DNA by polymerase chain reaction (PCR) amplification. Sperm could be squeezed out from a few F1 of transgenic allotetraploid fish of 150 days, however, this phenomenon did not exist in the controls. The importance of forming the pure line of transgenic allotetraploid was elucidate in the paper.