Journal of Genetics and Genomics

“Micro-deletions” of the human Y chromosome and their relationship with male infertility

Zheng Li, Christopher J Haines, Yibing Han

2008, 35(4): 193-199. doi: 10.1016/S1673-8527(08)60027-2

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Abstract:
The Y chromosome evolves from an autochromosome and accumulates male-related genes including sex-determining region of Y-chromosome (SRY) and several spermatogenesis-related genes. The human Y chromosome (60 Mb long) is largely composed of repetitive sequences that give it a heterochromatic appearance, and it consists of pseudoautosomal, euchromatic, and heterochromatic regions. Located on the two extremities of the Y chromosome, pseudoautosomal regions 1 and 2 (PAR1 and PAR2, 2.6 Mb and 320 bp long, respectively) are homologs with the termini of the X chromosome. The euchromatic region and some of the repeat-rich heterochromatic parts of the Y chromosome are called “male-specific Y” (MSY), which occupy more than 95% of the whole Y chromosome. After evolution, the Y chromosome becomes the smallest in size with the least number of genes but with the most number of copies of genes that are mostly spermatogenesis-related. The Y chromosome is characterized by highly repetitive sequences (including direct repeats, inverted repeats, and palindromes) and high polymorphism. Several gene rearrangements on the Y chromosome occur during evolution owing to its specific gene structure. The consequences of such rearrangements are not only loss but also gain of specific genes. One hundred and fifty three haplotypes have been discovered in the human Y chromosome. The structure of the Y chromosome in the GenBank belongs to haplotype R1. There are 220 genes (104 coding genes, 111 pseudogenes, and 5 other uncategorized genes) according to the most recent count. The 104 coding genes encode a total of about 48 proteins/protein families (including putative proteins/protein families). Among them, 16 gene products have been discovered in the azoospermia factor region (AZF) and are related to spermatogenesis. It has been discovered that one subset of gene rearrangements on the Y chromosome, “micro-deletions”, is a major cause of male infertility in some populations. However, controversies exist about different Y chromosome haplotypes. Six AZFs of the Y chromosome have been discovered including AZFa, AZFb, AZFc, and their combinations AZFbc, AZFabc, and partial AZFc called AZFc/gr/gr. Different deletions in AZF lead to different content spermatogenesis loss from teratozoospermia to infertility in different populations depending on their Y haplotypes. This article describes the structure of the human Y chromosome and investigates the causes of micro-deletions and their relationship with male infertility from the view of chromosome evolution. After analysis of the relationship between AZFc and male infertility, we concluded that spermatogenesis is controlled by a network of genes, which may locate on the Y chromosome, the autochromosomes, or even on the X chromosome. Further investigation of the molecular mechanisms underlying male fertility/infertility will facilitate our knowledge of functional genomics.

Development of microsatellite markers and their utilization in genetic diversity analysis of cultivated and wild populations of the mud carp (Cirrhina molitorella)

Cheng Yang, Xinping Zhu, Xiaowen Sun

2008, 35(4): 201-206. doi: 10.1016/S1673-8527(08)60028-4

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Abstract:
Microsatellite markers have been increasingly used in genetic studies on fishery species because of their high applicability in selective breeding programs. Here we reported the development of microsatellite markers and their utilization in mud carp (Cirrhina molitorella). An (CA)₁₅ enriched library has been constructed for mud carp, using the magnetic beads enrichment procedure. Sequence analysis of 60 randomly picked positive colonies indicate that 56 (93.3%) of the colonies contain microsatellites. Microsatellite polymorphism was assessed using 10 mud carp individuals, and 12 microsatellite loci turned out to be polymorphic. We utilized these loci to study the genetic diversity of a wild population (WM) and a cultured population (CM) of the mud carp. A total of 109 alleles were detected with an average of 9.08 alleles per locus. The mean value of the observed heterozygosity of WM and CM was 0.6361 and 0.6417, respectively, and significant decrease of genetic diversity in CM was not observed. The genetic distance between the two populations was 0.1546 and the value of was 0.0473. This showed that there existed a slight genetic differentiation between WM and CM.

Identification and isolation of Mu-flanking fragments from maize

Yijun Wang, Guangming Yin, Qin Yang, Jihua Tang, Xiaomin Lu, Schuyler S. Korban, Mingliang Xu

2008, 35(4): 207-213. doi: 10.1016/S1673-8527(08)60029-6

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Abstract:
Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics. Isolation of genomic segments flanking the insertionMutator (Mu) is a key step in insertion mutagenesis studies. Herein, we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize. The method consists of the following steps: 1) double-digestion of genomic DNA with Bgl II/Msp I and ligation of digested fragments to the Bgl II- and Msp I-adaptors; 2) enrichment of a subset of Bgl II/Msp I fragments followed by selective amplification of the Mu-flanking fragments; 3) simultaneous display of AFLP bands derived from the flanking regions for both insert and native Mu transposons; 4) identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5) confirmation of flanking fragments related to these Mu insertions. Using this approach, we have isolated flanking fragment(s) resulting from Mu insertion for every Mu-induced mutant, and one such fragment, M196-FF, is found to contain a partial sequence of the DNA topoisomerase I gene Top1. Moreover, the modified AFLP method including all restriction enzymes, adaptors and primers has been optimized in this study. The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.

Transcriptional regulation of human polo-like kinases and early mitotic inhibitor

Moe Tategu, Hiroki Nakagawa, Kaori Sasaki, Rieko Yamauchi, Sota Sekimachi, Yuka Suita, Naoko Watanabe, Kenichi Yoshid

2008, 35(4): 215-224. doi: 10.1016/S1673-8527(08)60030-2

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Abstract:
Human polo-like kinases (PLK1–PLK4) have been implicated in mitotic regulation and carcinogenesis. PLK1 phosphorylates early mitotic inhibitor 1 (Emi1) to ensure mitosis entry, whereas Emi2 plays a key role during the meiotic cell cycle. Transcription factor E2F is primarily considered to regulate the G₁/S transition of the cell cycle but its involvement in the regulation of mitosis has also been recently suggested. A gap still exists between the molecular basis of E2F and mitotic regulation. The present study was designed to characterize the transcriptional regulation of human PLK and Emi genes. Adenoviral overexpression of E2F1 increased PLK1 and PLK3 mRNA levels in A549 cells. A reporter gene assay revealed that the putative promoter regions of PLK1, PLK3, and PLK4 genes were responsive to activators E2F, E2F1–E2F3. We further characterized the putative promoter regions of Emi1 and Emi2 genes, and these could be regulated by activators E2F and E2F1–E2F4, respectively. Finally, PLK1–PLK4, Emi1, and Emi2 mRNA expression levels in human adult, fetal tissues, and several cell lines indicated that each gene has a unique expression pattern but is uniquely expressed in common tissues and cells such as the testes and thymus. Collectively, these results indicate that E2F can integrate G₁/S and G₂/M to oscillate the cell cycle by regulating mitotic genes PLK and Emi, leading to determination of the cell fate.

Genetic polymorphism of mitochondrial DNA HVS-I and HVS-II of Chinese Tu ethnic minority group

Feng Chen, Yajun Deng, Yonghui Dang, Bo Zhang, Haofang Mu, Xiaoguang Yu, Lin Li, Chunxia Yan, Teng Chen

2008, 35(4): 225-232. doi: 10.1016/S1673-8527(08)60031-4

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Abstract:
We analyzed the two hypervariable segments HVS-I and HVS-II of 108 Chinese Tu ethnic minority group samples for forensic and population genetics purposes. Comparing with Anderson sequence, 79 polymorphic loci in HVS-I and 40 in HVS-II were found in Chinese Tu ethnic minority group mtDNA sequences, and 90 and 64 haplotypes were then defined. Haplotype diversity and the mean pairwise differences were 0.9903±0.0013 and 5.7785 in HVS-I, and 0.9777±0.0013 and 3.5819 in HVS-II, respectively. By analyzing the hypervariable domain from nucleotide 1,6180 to 1,6193 in HVS-I, we defined some new types of sequence variations. We also compared the relationship between Tu population and other populations using mtDNA HVS-I sequences. According to Rst genetic distances, the phylogenetic tree showed that the Tu population, the Xi'an Han population, the Chinese Korean, and the Mongol ethnic group were in a clade. This indicated a close genetic relationship between them. There were far relations between the Tu population and other Chinese southern Han populations, Siberian, European, African, and other foreign populations. The results suggest that Tu population has a multi-origin and has also merged with other local populations.

Analysis of genetic diversity and population structure of Chinese yak breeds (Bos grunniens) using microsatellite markers

Guixiang Zhang, Weisheng Chen, Ming Xue, Zhigang Wang, Hong Chang, Xu Han, Xinjun Liao, Donglei Wang

2008, 35(4): 233-238. doi: 10.1016/S1673-8527(08)60032-6

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Abstract:
Nine Chinese yak breeds (Maiwa, Tianzhu White, Qinghai Plateau, Sibu, Zhongdian, Pali, Tibetan High Mountain, Jiulong, and Xinjiang) and Gayal were analyzed by means of 16 microsatellite markers to determine the level of genetic variation within populations, genetic relationship between populations, and population structure for each breed. A total of 206 microsatellite alleles were observed. Mean F-statistics (0.056) for 9 yak breeds indicated that 94.4% of the genetic variation was observed within yak breeds and 5.6% of the genetic variation existed amongst breeds. The Neighbor-Joining phylogenetic tree was constructed based on Nei's standard genetic distances and two clusters were obtained. The Gayal separated from the yaks far away and formed one cluster and 9 yak breeds were grouped together. The analysis of population structure for 9 yak breeds and the Gayal showed that they resulted in four clusters; one cluster includes yaks from Tibet Autonomous Region and Qinghai Province, one cluster combines Zhongdian, Maiwa, and Tianzhu White, and Jiulong and Xinjiang come into the third cluster. Pali was mainly in the first cluster (90%), Jiulong was mainly in the second cluster (87.1%), Zhongdian was primarily in the third cluster (83%), and the other yak breeds were distributed in two to three clusters. The Gayal was positively left in the fourth cluster (99.3%).

Expression of non-structural protein NS3 gene of Bombyx mori densovirus (China isolate)

Huijuan Yin, Qin Yao, Zhongjian Guo, Fang Bao, Wei Yu, Jun Li, Keping Chen

2008, 35(4): 239-244. doi: 10.1016/S1673-8527(08)60033-8

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Abstract:
The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 (China isolate), named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects of the NS3 gene during viral DNA replication, a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus (China isolate). Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe, respectively. The NS3 protein was expressed inEscherichia coli BL21. The pFastBacHTe-NS3 was transformed to E. coli DH10Bac. The recombinant bacmid baculoviruses (rBacmid-EGFP-NS3) isolated from the white colonies were transfected into BmN-4 cells using a transfection reagent. BmN-4 cells were infected with recombinant virus to express fusion proteins. The expression of fusion protein around 30 kDa in E. coli BL21 was identified by SDS-PAGE, Western blotting, and mass spectrometry. The expressed NS3 protein by B. mori nucleopolyhedrovirus bacmid system was confirmed by Western blotting using an anti-NS3 polyclonal antibody. And about 45 kDa protein was found. The expressed fusion protein was smaller than the expected size of EGFP-NS3, 55 kDa. Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected larvae with smaller molecular size.

Construction and analysis of a plant transformation binary vector pBDGG harboring a bi-directional promoter fusing dual visible reporter genes

Chunxiao Zhang, Ying Gai, Wenqi Wang, Yanyan Zhu, Xuemei Chen, Xiangning Jiang

2008, 35(4): 245-249. doi: 10.1016/S1673-8527(08)60034-X

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Abstract:
The constitutive promoter of cauliflower mosaic virus 35S (CaMV 35S) is a polar unidirectional promoter and is widely used in plant genetic engineering. In the present study, the unidirectional CaMV 35S promoter has been modified to a bi-directional promoter by fusing its minimal promoter element to the 5′ end of CaMV 35S promoter in the opposite orientation. To qualitatively and quantitatively estimate its bi-directional transcriptional function and activity, two visible reporter genes,gusA (β-glucuronidase, GUS) and gfp (green fluorescent protein, GFP), were fused to the two ends of the promoter in bi-orientations ending with NOS terminator sequences, respectively. Stable expression of gusA and gfp genes in transgenic tobacco (Nicotiana tabacum L.) was visulized by histochemically staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants. The expression of two reporter genes showed that the constructed bi-directional promoter did have the bi-directional transcriptional function in both expected orientations. The quantitative estimation of GUS and GFP were determined on a HITACHI F1000 Fluorescence Spectrophotometer with various wavelengths of excitation and emission. The GUS activity varied from 8 to 250 pmol 4-MU / min / mg protein and the GFP content varied from 0.9 to 1.8 μg/mg protein in various lines of transgenic tobacco plants. Higher GUS activity generally coupled with lower GFP content, and vice versa.

Identification and characterization of cDNA sequences encoding the HIS3 and LEU2 genes of the fungus Alternaria tenuissima

Ying Wan, Xuli Wang, Yun Huang, Dewen Qiu, Linghuo Jiang

2008, 35(4): 251-256. doi: 10.1016/S1673-8527(08)60035-1

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Abstract:
Alternaria tenuissima is a fungus widely present in the environment and could cause diseases in plants and humans. In this study, through a yeast genetic approach, cDNA sequences were isolated and characterized for the AtHIS3 and AtLEU2 genes. AtHIS3 cDNA encodes a protein of 238 amino acids, while AtLEU2 cDNA encodes a protein of 363 amino acids. Based on the phylogenetic analysis of amino acid sequences of AtHis3p and AtLeu2p, A. tenuissima is closely related to the plant pathogenic fungus Phaeosphaeria nodorum. This study provides two genetic markers for studies of functions of genes regulating development, morphology, and virulence ofA. tenuissima.

2008 Vol. 35, No. 4