Karyotypic evolution of a novel cervid satellite DNA family isolated by microdissection from the Indian muntjac Y-chromosome

Abstract

A minilibrary was constructed from DOP-PCR products using microdissected Y-chromosomes of Indian muntjac as DNA templates. Two microclones designated as IM-Y4-52 and IM-Y5-7 were obtained from negative screening of all three cervid satellite DNAs (satellites I, II, and IV). These two microclones were 295 and 382 bp in size, respectively, and shared approximately 70% sequence homology. Southern blot analysis showed that the IM-Y4-52 clone was repetitive in nature with an approximately 0.32-kb register in HaeIII digest. Sequence comparison revealed no similarities to DNA sequences deposited in the GenBank database, suggesting that the microclone sequences were from a novel satellite DNA family designated as cervid satellite V. A subclone of an Indian muntjac BAC clone which screened positive for IM-Y4-52 had a 3,325-bp insert containing six intact monomers, four deleted monomers, and two partial monomers. The consensus sequence of the monomer was 328 bp in length and shared more than 80% sequence homology with every intact monomer. A zoo blot study using IM-Y4-52 as a probe showed that the strong hybridization with EcoRI digested male genomic DNA of Indian muntjac, Formosan muntjac, Chinese muntjac, sambar deer, and Chinese water deer. Female genomic DNA of Indian muntjac, Chinese water deer, and Formosan muntjac also showed positive hybridization patterns. Satellite V was found to specifically localize to the Y heterochromatin region of the muntjacs, sambar deer, and Chinese water deer and to chromosome 3 of Indian muntjac and the X-chromosome of Chinese water deer.

Centromere Evolution: Digging into Mammalian Primary Constriction

“…In two evolutionarily distant mammals, the marsupial Macropus rufogriseus and the rodent Microtus chrotorrhinus, giant sex chromosomes derived from a large block of heterochromatin at the centromeric and pericentromeric regions have been observed. Finally, evidence of a different rate at which autosomes and sex chromosomes accumulate and dissipate centromeric material has been found in cervid deer, muntjac, and in the genus Macropus (Bulazel et al, 2006;Li et al, 2005;Lin & Li, 2006), with the retention for longer periods of time of tandem arrays of ancestral satellites in the sex chromosomes that are not found in the autosomes.…”

Organization and evolution of a novel cervid satellite DNA with yeast CDEI-like repeats

“…Cell lines, chromosome preparations, and DNA isolation Metaphase chromosome spreads and genomic DNAs were prepared from the fibroblast cell lines of the following species: Indian muntjac (Muntiacus muntjak vaginalis) (male cell line, CCL-157, American Type Culture Collection; female cell line, kindly supplied by Dr. Andrew P. Feinberg, School of Medicine, Johns Hopkins University, Baltimore, MD, USA); Formosan muntjac (M. reevesi micrurus) ; caribou (Rangifer tarandus caribou) (Lin et al 1991;Lee et al 1994;Lin et al 2004); male black-tailed deer (Odocoileus hemionus hemionus) (CRL-6193, American Type Culture Collection); female Chinese water deer (Hydropotes inermis) (kindly provided by Dr. F. Yang, Centre for Veterinary Science, University of Cambridge, Cambridge, UK); Roe deer (Capreolus capreolus capreolus) cell line (from San Diego Zoo, San Diego, CA, USA) and male Formosan sambar deer (Cervus unicolor swinhoei) (Li et al 2005). Metaphase chromosome spreads and genomic DNAs of Formosan sika (Cervus nippon taioanus) and Asian red deer (Cervus elaphus) were prepared from blood samples provided by a certified deer farm in Taiwan.…”

“…The transformation mixture was plated onto Luria-Bertani (LB) agar plates containing 100 μg/ml of kanamycin, 40 μg/ml of X-gal, and 0.05 mmol/l IPTG to construct a X + 3 centromere mini-library (designated as the pIMCentX + 3). The detailed protocol used for chromosome microdissection and DOP-PCR was referred to Li et al (2005).…”

“…All duplicated microclones were lifted onto nylon membranes and denatured in an alkali solution. A probe mixture containing 32 P-labeled satellite I (C5; (Lin et al 1991)), satellite II (Mmv-0.7; (Li et al 2000b)) , satellite IV (Mmv-1.0; (Li et al 2002)), and satellite V (Mmv-0.32#1; (Li et al 2005)) was used to hybridize the membranes for the first screening. A 32 P-labeled DOP-PCR amplified X + 3-microdissected DNA probe was used to hybridize the membranes for the second screening.…”

Estudos citogenéticos no genêro Characidium (Teleostei, Characiformes, Chrenuchidae), com análise estrutural e molecular do sistema ZZ-ZW de cromossomos sexuais

“…Em humanos, esta técnica, aliada às metodologias de citogenética clássica tem sido uma poderosa ferramenta da genética clínica na caracterização de complexos rearranjos cromossômicos (Meltzer et al, 1992;Wiltshire et al, 2001;Nuntakarn et al, 2002;Hu et al, 2004). No contexto da evolução cromossômica, a metodologia de micro-FISH tem sido utilizada na identificação dos rearranjos cromossômicos envolvidos no processo evolutivo de primatas (Neusser et al, 2005), cervídeos (Li et al, 2005), roedores (Marchal et al,. 2004) e insetos , entre outros.…”