Construction of a smallMus musculusrepetitive DNA library: identification of a new satedllite sequence inMus musculus

Abstract

We report the construction of a small library of recombinant plasmids containing Mus musculus repetitive DNA inserts. The repetitive cloned fraction was derived from denatured genomic DNA by reassociation to a Cot value at which repetitive, but not unique, sequences have reannealed followed by exhaustive S1 nuclease treatment to degrade single stranded DNA. Initial characterizations of this library by colony filter hybridizations have led to the identification of a previously undetected M. musculus minor satellite as well as to clones containing M. musculus major satellite sequences. This new satellite is repeated 10-20 times less than the major satellite in the M. musculus genome. It has a repeat length of 130 nucleotides compared with the M. musculus major satellite with a repeat length of 234 nucleotides. Sequence analysis of the minor satellite has shown that it has a 29 base pair region with extensive homology to one of the major satellite repeating subunits. We also show by in situ hybridization that this minor satellite sequence is located at the centromeres and possibly the arms of at least half the M musculus chromosomes. Sequences related to the minor satellite have been found in the DNA of a related Mus species, Mus spretus, and may represent the major satellite of that species.

Centromere Protein B of African Green Monkey Cells: Gene Structure, Cellular Expression, and Centromeric Localization

“…The ␣21-I array colocalized with the antigenic sites of ACA staining in both the interphase nucleus and metaphase chromosomes. CENP-B boxes (a 16-of-17-bp match to the human consensus) have been found in mouse (Mus musculus) minor satellite DNA (24), which is similarly located at the primary constrictions of metaphase chromosomes and composed of 120-bp repeat units whose nucleotide sequence is unrelated to that of ␣-satellite DNA except for the CENP-B box (31,49). Highly homologous cDNAs encoding CENP-B have been cloned from human and mouse (M. musculus) cells (10,38).…”

Most highly repeated dispersed DNA families in the mouse genome.

“…Therefore, we and others (52) believe that there exists a need to define the repertoire of repetitive DNA families in a mammal. Toward this aim and also with the intent of looking for regulatory elements and mammalian transposons, we have constructed a small library (125 insert- containing clones) of repetitive genomic DNA (48). This library, being randomly generated, is representative of the entire genome and contains several cloned sequences corresponding to each of the most repeated (>30,000 copies) DNA families.…”

“…The original library of genomic repetitive sequences was constructed at the Pstl site of pBR322. Repetitive hybrids were cloned with the use of GC tails (48). The insert-containing plasmids have been maintained in the Escherichia coli strain X1776.…”

“…RESULTS Construction of a repetitive sequence library and identification of clones corresponding to the most repeated sequence families. The method of library construction is described in detail elsewhere (48) and is similar to that used by Scheller and colleagues for the sea urchin (50). Briefly, genomic DNA was partially restricted with the enzyme HaeII to an average size of 5 kb and annealed under conditions (Cot, 0.03) such that all foldback and 95% of the major satellite DNA was reassociated.…”

“…Ten clones produced the characteristic ladder of the mouse major satellite when used to probe DNA restricted with EcoRII and other enzymes (data not shown). One clone corresponds to a newly identified mouse minor satellite (48). Six additional clones were identified as MIF-1 sequences which hybridize to a characteristic and highly conserved 1.3-kb EcoRI band or to characteristic MspI bands (see Fig.…”