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Biochemical Genetics (v.47, #3-4)

Mitochondrial DNA Sequence Variation of the Swallowtail Butterfly, Papilio xuthus, and the Cabbage Butterfly, Pieris rapae by Heon Cheon Jeong; Jung A. Kim; Hyun Hwak Im; Hyung Uk Jeong; Mee Yeon Hong; Jong Eun Lee; Yeon Soo Han; Iksoo Kim (pp. 165-178).

We analyzed a portion of mitochondrial COI gene sequences (658 bp) to investigate the genetic diversity and geographic variation of the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae), and the cabbage butterfly, Pieris rapae L. (Lepidoptera: Pieridae). Papilio xuthus showed a moderate level of sequence divergence (0.91% at maximum) in 15 haplotypes, whereas Pi. rapae showed a moderate to high level of sequence divergence (1.67% at maximum) in 30 haplotypes, compared with other relevant studies. Analyses of population genetic structure showed that most populations are not genetically differentiated in both species. The distribution pattern of both species appears to be consistent with category IV of the phylogeographic pattern sensu Avise: a phylogenetic continuity, an absence of regional isolation of mtDNA clones, and extensive distribution of close clones. The observed pattern of genetic diversity and geographic variation of the two butterfly species seem to reflect the abundant habitats, abundant host plants, and flying abilities in connection with the lack of historical biogeographic barriers.

Keywords: Mitochondrial DNA; COI gene; DNA barcode; Papilio xuthus ; Pieris rapae ; Population genetic structure; Genetic diversity; Geographic variation

Cloning and Characterization of the SSB-1 and SSB-4 Genes Expressed in Zebrafish Gonads by Jian-Zhen Li; Ya-Ping Zhou; Yan Zhen; Yan Xu; Peng-Xiang Cheng; Huan-Nan Wang; Feng-Jiao Deng (pp. 179-190).

The protein of the gustavus (gus) gene has a typical SOCS box domain and repeats in the splA and RyR (SPRY) domains. GUS can interact with Vasa and is necessary for the specification of germ cells. We cloned two zebrafish genes, SSB-1 and SSB-4 (SPRY domain SOCS box proteins). Phylogenetic analysis shows that zebrafish SSB-1 and SSB-4 are clustered into clades of SSB-1-like and SSB-4-like genes from other species. RT-PCR analysis of tissues revealed that zebrafish SSB-1 and -4 are expressed in the ovary and testis. We investigated the spatial expression patterns of zebrafish SSB-1 and -4 in embryos from the two-cell stage to 72 h postfertilization (hpf) using whole-mount in situ hybridization. SSB-1 and -4 transcripts were present in all blastomeres during the early embryonic stages, but the genes differ in their expression pattern. SSB-4 mRNA was located in the region of the primordial germ cells in 24 and 72 hpf embryos, but SSB-1 mRNA was not detected at these stages. We hypothesize that SSB-4 plays a role in the early development of germ cells.

Keywords: Zebrafish; SSB-1 ; SSB-4 ; Gus ; Primordial germ cells

Sequence Analysis of the Plasmid pGY1 Harbored in Salmonella enterica Serovar Paratyphi A by H. Huang; J. Li; X. L. Yang; Y. G. Wang; Y. P. Wang; J. S. Tao; Y. Z. Huang; X. L. Zhang (pp. 191-197).

The cryptic plasmid pGY1, which is harbored by a clinical isolate of Salmonella enterica serovar Paratyphi A, was identified in a 9-year-old girl with paratyphoid fever in 2005, and its DNA sequence was determined. It is 3592 bp in length and had a G+C content of 43.3%. Three ORFs were predicted that share low similarity with hypothetical proteins in the GenBank database. pGY1 shared 36.6% sequence homology with the cryptic plasmid pIMVS1 from Salmonella typhimurium. Its unique sequence makes it attractive for further study to obtain insight into the evolutionary relationship of this plasmid with other Enterobacteriaceae plasmids.

Keywords: Salmonella enterica serovar Paratyphi A; Plasmid; Sequence

Characterization and SNP Identification of Part of the Goat Melanophilin Gene by Fu-Jun Feng; Xiang-Long Li; Rong-Yan Zhou; Gui-Ru Zheng; Lan-Hui Li; Dong-Feng Li (pp. 198-206).

The melanophilin (MLPH) gene has been characterized as the candidate gene for dilute coat color in some species, but little is known about it in the goat. In this study, part of the genomic DNA sequence (19,289 bp) containing the whole coding region of the MLPH gene from goat, as well as from sheep, was determined. We found 16 exons and 15 introns; the coding region was 1767 bp distributed in 15 exons (2–16). In sheep, the length of part of the genomic DNA sequence was 16,988 bp, with 16 exons and 15 introns, and the coding region was 1833 bp, distributed in 15 exons (2–16). Dozens of SNPs as well as some noticeable motifs in the goat MLPH gene were found during the process of sequencing and polymorphism screening. Based on the SSR Tool, three simple sequence repeat motifs were detected in the goat and sheep DNA sequences. Compared with cattle, we found insertions of 4 amino acids in goats and 26 amino acids in sheep.

Keywords: Melanophilin; Goat; Coding SNPs; Linkage

Positive Selection Drives the Evolution of Bat Bitter Taste Receptor Genes by Yingying Zhou; Dong Dong; Shuyi Zhang; Huabin Zhao (pp. 207-215).

Bitter taste reception is expected to be associated with dietary selection and to prevent animals from ingesting potentially harmful compounds. To investigate the genetic basis of bitter taste reception, we reconfirmed the bitter taste receptor (T2R) genes from cow (herbivore) and dog (carnivore) genome sequences and identified the T2R repertoire from the draft genome of the bat (insectivore) for the first time using an automatic data-mining method. We detected 28 bitter receptor genes from the bat genome, including 9 intact genes, 8 partial but putative functional genes, and 9 pseudogenes. In the phylogenetic analysis, most of the T2R genes from the three species intermingle across the tree, suggesting that some are conserved among mammals with different dietary preferences. Furthermore, one clade of bat-specific genes was detected, possibly implying that the insectivorous mammal could recognize some species-specific bitter tastants. Evolutionary analysis shows strong positive selection was imposed on this bat-specific cluster, indicating that positive selection drives the functional divergence and specialization of the bat bitter taste receptors to adapt diets to the external environment.

Keywords: Positive selection; Adaptive evolution; Genome; Bat; Diet; Bitter taste; T2R

Evaluation of Genetic Variation in the Clown Knifefish, Chitala chitala, Using Allozymes, RAPD, and Microsatellites by Anup Mandal; Kuldeep Kumar Lal; Vindhya Mohindra; Rajeev Kumar Singh; Peyush Punia; U. K. Chauhan; Wazir Singh Lakra (pp. 216-234).

Twenty-seven enzyme systems, six random amplified polymorphic DNA (RAPD) primers, and two microsatellite loci were tested to determine intraspecific divergence in the natural population of the endangered Indian featherback fish, Chitala chitala, for the first time. The 262 samples of C. chitala were collected from six riverine locations in India: the Satluj, Ganga (Ghagra, Bhagirathi, and Brahmaputra), Mahanadi, and Narmada river systems. The analysis revealed population subdivisions, with an F_ST value from 0.1235 (95% confidence 0.0868–0.1621) for RAPD and a combined F_ST of 0.0344 (95% confidence 0.0340–0.0350) for microsatellite loci. An analysis of 38 allozyme loci did not reveal any polymorphism in the samples from any of the riverine localities; a possible explanation for this could be that the ancestors of Chitala could have faced a population reduction in prehistoric periods, as low allozyme variation is also reported for other species of Chitala from south Asia.

Keywords: Chitala chitala ; Genetic diversity; Allozymes; Microsatellites; RAPD

Genetic Diversity of Red-Bellied Titis (Callicebus moloch) from Eastern Amazonia Based on Microsatellite Markers by Luciana Alcantarino Menescal; Evonnildo Costa Gonçalves; Artur Silva; Stephen Francis Ferrari; Maria Paula Cruz Schneider (pp. 235-240).

The titi monkeys (Callicebus spp.) are a large, diverse genus of platyrrhines, widely distributed in tropical South America. The genetic variability of these monkeys is still relatively poorly known, especially at the population level. In the present study, four heterologous microsatellite markers were used to investigate genetic diversity in 23 individuals from a wild population of red-bellied titis (Callicebus moloch) in eastern Amazonia. An unexpectedly low level of diversity was found. The average number of alleles was 8.75 (range: 5–15), and the average heterozygosity was 0.33 (range: 0.09–0.65). This preliminary information suggests a reduction of the potential for long-term survival of the population and indicates the putative necessity of implementation of a species conservation program.

Keywords: Callicebus moloch ; Population genetics; Microsatellites; Conservation

Genetic Variability, Differentiation, and Founder Effect in Golden Jackals (Canis aureus) from Serbia as Revealed by Mitochondrial DNA and Nuclear Microsatellite Loci by Frank E. Zachos; Dusko Cirovic; Julia Kirschning; Marthe Otto; Günther B. Hartl; Britt Petersen; Ann-Christin Honnen (pp. 241-250).

We analyzed 121 golden jackals (Canis aureus) from six sample sites in Serbia with regard to genetic variability and differentiation as revealed by mitochondrial control region sequences and eight nuclear microsatellite loci. There was no variation at all in the mtDNA sequences, and nuclear variability was very low (average observed and expected heterozygosity of 0.29 and 0.34, respectively). This is in line with the considerable recent range expansion of this species in the Balkans and indicates a strong founder effect in the recently established Serbian population. We did not find evidence of differentiation between the northeastern jackals and those from the plain of Srem or those in between. F-statistics and Bayesian Structure analyses, however, were indicative of a low degree of overall differentiation in the Serbian population. A vagrant Austrian jackal that was also analyzed was genetically indistinguishable from its Serbian conspecifics.

Keywords: Canis aureus ; Serbia; Founder effect; Microsatellites; Mitochondrial DNA

Novel Polymorphism at the 3′ UTR of the Caprine Adiponectin Gene by Xianyong Lan; Tianbao Wei; Liangzhi Zhang; Hong Chen; Shenrong Hu; Chuzhao Lei; Xing Tang Fang (pp. 251-256).

Molecular Cloning and Evolutionary Analysis of Hemoglobin α-Chain Genes in Bats by Yang Liu; Dong Dong; Nai-Jian Han; Hua-Bin Zhao; Jin-Shuo Zhang; Gang Li; Paul A. Racey; Shu-Yi Zhang (pp. 257-265).

Bats are the only mammals with the capacity for powered flight. When flying, they need abundant energy and oxygen. According to previous works, the hemoglobin (Hb) oxygen loading function of bats is insensitive to variations in body temperature, although different bat species have different heat sensitivity. We cloned Hb α-chain sequences from eight bat species to investigate whether they have different characteristics. We found that Hb in the bat lineages is under purifying selection, which accords with the importance of its function in bats. Three turn regions in bat Hb, however, have distinct evolutionary rates compared with those of other mammals, and the codons in these regions have an accelerated rate of evolution. These codons are under divergent selection in bats. These changes in Hb may have occurred in response to the physiological requirements of the species concerned, as adaptations to different lifestyles.

Keywords: Hemoglobin; α-chain; Gene; Bats; Evolution

Molecular Cloning, Expression, and Chromosomal Mapping of the Porcine CDC-2-Like Kinase 1 (CLK1) Gene by Shui Hua Xie; Jia Qi Li; Yao Sheng Chen; Ping Gao; Hao Zhang; Ze Yue Li (pp. 266-275).

CDC-2-like kinase 1 (CLK1) plays a critical role in regulating pre-RNA splicing and post-transcriptional gene expression. Two distinct transcripts of the porcine CLK1 gene, known as full-length CLK1 and truncated CLK1 (CLK1 T), were identified by in silico cloning, RT-PCR, and RACE. The entire cDNA sequence of full-length CLK1 was 1771 bp, containing a 1455 bp ORF encoding a deduced protein of 484 amino acids. The complete cDNA sequence of CLK1 T is 1680 bp, containing a 414 bp ORF encoding a deduced protein of 137 amino acids. The genomic structure and sequence of porcine CLK1 were analyzed using a bacterial artificial chromosome clone of a Chinese Erhualian pig, with 13 exons and 12 introns spanning approximately 9 kb. RT-PCR revealed that the full-length and truncated splice forms were expressed at equivalent levels in pig heart, fat, liver, spleen, and lymph tissues. The full-length splice form was expressed at a much higher level than the truncated form in tissues of the pig cerebrum, longissimus dorsi, small intestine, and kidney. The CLK1 gene was physically assigned to SSC 15 between microsatellite markers SW1316 and SW2083 using the IMpRH panels.

Keywords: Pig; STY; Dual specificity kinase; Radiation hybrid mapping; Alternative splicing

Genetic Diversity and Variation Among Botanical Varieties of Old Portuguese Wheat Cultivars Revealed by ISSR Assays by Ana Carvalho; José Lima-Brito; Benvindo Maçãs; Henrique Guedes-Pinto (pp. 276-294).

Inter-simple sequence repeats (ISSRs) were used for genetic diversity analyses of an Old Portuguese wheat collection. Eighteen primers produced 96.3 and 98.5% of ISSR polymorphism in bread and durum wheat cultivars, respectively. The unweighted pair group method with arithmetical averages (UPGMA) phenogram clearly split all cultivars based on their species/ploidy, reflecting a defined genetic structure. ISSRs revealed high genetic diversity at interspecific, intraspecific, and intercultivar levels. Thirty-three exclusive ISSR markers were found. Cultivars were clustered according to their botanical varieties and, in a few cases, with their homonym(s). No statistically significant differences were found between genetic diversity parameters of durum and bread wheat, probably due to high intraspecific diversity. Similar analyses were performed among botanical varieties, and their relationships were defined. Cladograms resembled UPGMA clustering. This highly genetically diverse Old wheat collection will be conserved and maintained, and it could be further used in breeding programs to widen the narrow genetic basis of modern wheat varieties and to avoid the loss of rare and unique alleles.

Keywords: ISSRs; Genetic diversity; Genetic variation; Portuguese wheat germplasm

Melanocortin-4 Receptor Gene Polymorphisms in Obese Patients by Erkan Yurtcu; Akin Yilmaz; Zubeyde Ozkurt; Emine Kolukisa; Murat Yilmaz; Hatice Keles; Mehmet Ali Ergun; Ilhan Yetkin; Adnan Menevse (pp. 295-300).

Obesity is a complex disease caused by both genetics and environmental factors. Melanocortin-4 receptor (MC4R) (MIM 155541) gene polymorphisms were reported to be the cause of monogenic obesity in humans. We studied three polymorphisms (Val50Met, Val103Ile, and Ser58Cys) and a mutation (Asn274Ser) of the MC4R gene in 203 obese patients and in 110 healthy subjects in the Turkish population. A high incidence of Val103Ile and Val50Met polymorphisms as well as the Asn274Ser mutation was found in the obese patients, whereas no significant correlation was found regarding the Ser58Cys polymorphism. We conclude that there is a concordance between the polymorphisms (Val103Ile, Val50Met, Ser58Cys) that were first studied in the Turkish population with obesity.

Keywords: MC4R; Obesity; PCR-RFLP

Isolation and Characteristics of the CN Gene, a Tobacco Mosaic Virus Resistance N Gene Homolog, from Tobacco by Gai-Yun Zhang; Ming Chen; Jia-Ming Guo; Tong-Wen Xu; Lian-Cheng Li; Zhao-Shi Xu; You-Zhi Ma; Xue-Ping Chen (pp. 301-314).

Nicotiana rustica L. HZNH, a native Chinese tobacco germplasm, displays a hypersensitive response (HR) and systemic acquired resistance following infection with tobacco mosaic virus (TMV). A resistance gene, CN, cloned from HZNH plants, was homologous to the N and NH genes identified in other Nicotiana species. The CN coding region (3423 bp) shares 93.63% and 86.50% nucleotide identity with N and NH, respectively. Whereas the five CN exon sequences are highly homologous with those of N and NH, the four introns differ significantly in length and sequence. Sequence analysis revealed that CN belongs to the TIR/NBS/LRR gene class. Expression of CN was up-regulated after TMV infection and was temperature sensitive. Organ-specific expression analysis suggested that CN transcripts accumulated at high levels in leaves, low levels in stems, and minimal levels in roots. When CN was inserted into TMV-susceptible N. tabacum cv. K326 plants by Agrobacterium-mediated transformation, the transgenic plants displayed HR and systemic HR due to uninhibited movement of the virus.

Keywords: Hypersensitive response; Systemic acquired resistance; TMV; Tobacco

Genetic Variation and Association of Insulin-Like Growth Factor Binding Protein-3 with Performance in Swine by Wenjun Wang; Qingyong Meng; Xiaoxiang Hu; Ning Li (pp. 315-321).

Genetic variation of insulin-like growth factor binding protein-3 was analyzed in 17 pig breeds (14 native Chinese and 3 European). Using PCR-single strand conformation polymorphism, we found a polymorphism in intron 2, and this SNP was the combined mutation of G897T-G903A-C911T. The Chinese breeds carried a higher TAT/TAT genotype frequency (over 50%), except for Bamei (22%), Yujiang Black (0.0%), and Erhualian (10.0%); the European breeds had a higher GGC/GGC genotype frequency (Large White 1.67%, Landrace 13.89%, Duroc 0.0%). The allelic frequency of TAT in Chinese breeds was over 50%, except for Yujiang Black (12.5%); the allelic frequency of GGC was over 50% in all European breeds. The effect of genotype on 43 performance traits was investigated in one population (Lantang × Landrace). Pigs with the TAT/TAT genotype had higher B-point and C-point back-fat thickness than pigs with the GGC/GGC genotype. The TAT/TAT pigs also scored higher in meat color than the GGC/GGC pigs. These results implied that IGFBP-3 may affect meat quality and carcass traits.

Keywords: Insulin-like growth factor binding protein-3; SNP; Performance; Swine

Erythrocyte Total Carbonic Anhydrase Esterase Activity in African American Obese Children: Reduction Starts at a Young Age by Adriana J. Malheiro; Kanwal K. Gambhir; Gail N. Bland; Cynthia K. Abrams; Verle Headings; M. Abdul-Rahman (pp. 322-328).

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Biochemical Genetics (v.47, #3-4)