Isolation and identification of a novel satellite DNA family highly conserved in several Cervidae species

Abstract

In an attempt to amplify cervid satellite II DNA from the genomes of Indian muntjac and Chinese muntjac, a pair of primers derived from the white tailed deer satellite II DNA clone (OvDII) yielded a prominent 1 kb polymerase chain reaction (PCR) product (in addition to the expected 0.7 kb satellite II DNA fragments) in both species. The ~1 kb products were cloned, sequenced, and analyzed by Southern blotting and fluorescence in situ hybridization (FISH). This revealed that thẽ 1 kb cloned sequences indeed represent a previously unknown cervid satellite DNA family, which is now designated as cervid satellite IV DNA. Approximately 1 kb PCR clones were also obtained from the genomes of the black tailed deer and Canadian woodland caribou with similar primer pairs. Extremely high sequence conservation (over 90% homology) was observed among the clones generated from all four deer species and PCRSouthern hybridization experiments further verified the co-amplification of two kinds of satellite DNA sequences with the same pair of primers. This satellite DNA was found to co-localize with centromeric proteins at the kinetochore by a simultaneous FISH and immunofluorescence study. Due to its high sequence conservation and close association with kinetochores, the newly identified satellite DNA may have a functional centromeric role.

Cloning, Characterization, and FISH Mapping of Four Satellite DNAs from Black Muntjac (<I>Muntiacus crinifrons</I>)and Fea's Muntjac (<I>M. feae</I>)

“…BM1.1k had a length of 1.1-kbp with 44.27% GC content. Multiple-alignment was performed among BM1.1k, MMV-1.0 (M. muntjak vaginalis satellite IV), MR-1.0 (M. reevesi satellite IV) (Li et al, 2002), and FM-sat IV (M. reevesi micrurus satellite IV) (Lin et al, 2004), 98.17% homology was found among these clones (Fig. 2e), suggesting that BM1.1k belongs to the satellite IV DNA family.…”

“…The products were fractionated on a 1.5% agarose gel. Three bands of 0.7, 1.1 and 1.4 kb are detected in these species; b: Schematic illustration of sequence comparison between M. crinifrons satellite II DNA clone (BM700) and Formosan muntjac satellite II clone (FM-satII); c: Schematic diagram DNA sequences comparison between M. feae satellite DNA II clone (FM700) and Formosan muntjac satellite II clone (FM-satII); d: Multiple-alignment of M. crinifrons satellite II clone (BM700), M. feae satellite II clone (FM700) and M. muntjak vaginalis satellite II clone (MMV-0.7) shows 85.69% homology (identical nucleotide sequences from these clones are shown in black boxes); e: Multiple-alignment of M. crinifrons satellite IV clone (BM1.1k), MMV-1.0 (M. muntjak vaginalis satellite IV) (Li et al, 2002), MR-1.0 (M. reevesi satellite IV) (Li et al, 2002), and FM-sat IV (M. reevesi micrurus satellite IV) (Lin et al, 2004) shows 98.17% similarity.…”

“…Predominant signals were observed at the centric regions of all chromosomes except for the Y chromosome which is relatively weaker, but non-random signals were also found at some interstitial locations along the chromosomes of M. crinifrons, with seven interstitial signals on Chr1, a: Electrophoretic analysis of PCR products. PCR products were amplified with a pair of Satellite II primers (Li et al, 2000b(Li et al, , 2002) from three muntjac species (IM, Muntiacus muntjak vaginalis; BM, M. crinifrons; Feas, M. feae) and 'con' stands for negative control without template. The products were fractionated on a 1.5% agarose gel.…”

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

“…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.…”

“…The centromeric/pericentromeric region of the Indian muntjac (Muntiacus muntjak vaginalis; barking deer) chromosome X + 3 is known to be exceptionally huge and compound in nature (Brinkley et al 1984) and may be composed of many different satellite DNA families (including functional centromeric and nonfunctional centromeric satellite DNAs as well as new and old satellite DNAs). Indeed, cervid satellite I (an old cervid satellite DNA) (Lee et al 1994;Lin et al 1991), cervid satellite II (a functional centromeric satellite DNA) (Vafa et al 1999;Li et al 2000b), and cervid satellite IV (Li et al 2002) had been found in the X + 3 centromere region of Indian muntjac.…”