The efficiency of simian virus 40 (SV40) DNA replication is dependent on the structural organization of the regulatory region. The enhancing effect of the G+C-rich 21-base-pair (bp) repeats on SV40 DNA replication is position and dose dependent and to some extent orientation dependent. The inverted orientation is about 50% as effective as the normal orientation of the 21-bp repeat region. Movement of the 21-bp The double-stranded circular genome of simian virus 40 (SV40) represents a simple model system for studying mechanisms involved in eucaryotic DNA replication (for a review, see reference 8). Replication of the 5,243-base-pair (bp) genome of SV40 begins at a unique site, and the elongation of nascent DNA chains proceeds bidirectionally (9, 14). Viral DNA replication depends mainly on cellular mechanisms. The analysis of naturally arising evolutionary variants and deletion mutants has narrowed the limits of the origin signal for SV40 DNA replication (core ori) to a segment of approximately 65 bp from 0.653 to 0.666 map units including a 27-bp palindrome and an A+T-rich sequence (5,11,12,22,32,42,48). Single nucleotide changes or small deletions within this segment abolish or alter replication (20,42,46). The in vivo initiation sites for DNA synthesis have been mapped within the ori segment in the 27-bp palindrome as well as within the three 21-bp, G+C-rich tandem repeats (25). The 21-bp repeat has an enhancing effect on SV40 DNA replication (5,15,28) and is a bidirectional promoter element for SV40 transcription (3,13,18,40,54). The 72-bp repeat is a position-and orientationindependent enhancer for transcription (2,4,15,16,21,41,55).We examined the effects of the orientation, location, and number of copies of the 21-bp repeat on SV40 DNA replication. The role(s) of the 72-bp repeat in SV40 DNA replication was also investigated. Fragments containing the SV40 ori sequence plus various portions of the normal or relocated regulatory sequences were cloned into bacterial plasmids. By mixed transfection of monkey cells and Southern blot analysis of newly replicated DNAs, we compared the effects of various structural rearrangements of the 21-or 72-bp repeat or both on the replication efficiency of the SV40 ori region. MATERIALS AND METHODSCells and plasmid vector. The BSC-1 line of African green monkey kidney cells was grown in 8% calf serum plus 2% * Corresponding author.fetal calf serum and Eagle minimal essential medium (MEM-10). The COS-1 line of African green monkey kidney cells (19) was grown in 5% fetal calf serum (MEM-5).The plasmid vector pKP45 (kindly provided by K. Peden, The Johns Hopkins University, School of Medicine) is derived from pBR322 by deletion of nucleotides 676 to 2364 which removes the sequence that is "poisonous" for plasmid replication in mammalian cells (23,36,43).Restriction endonuclease digestion and DNA fragment purification. Restriction endonucleases were purchased from New England BioLabs, Inc. (Beverly, Mass.) or Boehringer Mannheim Biochemicals (Indianapolis, Ind.) and used as su...

The construction of a small library of mouse repetitive DNA has been previously reported (Pietras et al., Nucleic Acids Res. 11:6965-6983, 1983). Here we report that the 35 plasmids in this library corresponding to highly repeated (>30,000 copies per genome) dispersed DNA sequences can be grouped into no more than 5 distinct families. These families together comprise 8 to 10% of the mouse genome. They include the previously described small elements Bi, B2, and R and the large MIF-1 element. Twelve of the 35 clones contain evolutionarily conserved (EC) sequences. One EC clone in our library mostly consists of alternating dCdT residues; another consists of tandem repeats of the sequence CCTCT. The majority of Bls and B2s in the genome appear to be homogeneous, whereas R sequences, ECs, and MIF-is are heterogeneous. Two earlier reports showed highly repeated mammalian DNA sequences in the herpesvirus genome (Peden et al., Cell 31:71-80, 1982; Puga et al., Cell 31:81-87, 1982). We show that sequences homologous to our EC clones are present in the herpesvirus genome, although these polypyrimidine stretches are not detected in poxvirus, adenovirus, and simian virus 40 genomes. We detect transcripts containing homology to all of these sequences in a nuclear transcription assay. Also, we show that small, polyadenylated RNA molecules homologous to B2 sequences are expressed in undifferentiated embryonal carcinoma cells but not in their differentiated derivatives. The significance of these findings is discussed.Several highly repeated, nonsatellite families of DNA sequences have been reported in the mouse (51). These include the following: Bi, which is the mouse Alu (30) equivalent (32); B2 (33); MIF-1, the mouse interspersed fragment, originally called the 1.3-kilobase (kb) EcoRI fragment but now known to be greater than 5 kb in its entirety (13,14,26,42); the R sequence, a 400-to 500-base-pair (bp)-size family first located in multiple copies amidst the immunoglobulin cluster (20, 35); and EC1, an evolutionarily conserved (EC) sequence described in humans and mice (2, 41). EC1 is conserved in all eucaryotes, including yeast cells, so its hybridization to mammalian DNA is reduced considerably if unlabeled eucaryotic DNA is used as a carrier in the experiments.It is our prejudice that each repetitive sequence family will be found to have a very different role and history in the mouse genome, and as such, one cannot consider repetitive DNA in bulk in terms of functional versus selfish roles (17, 43). Therefore, we and others (52) 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. As our library should contain a representative of every family repeated more than 20,000 to 30,000 times, we have an opportunity to sort out how many highly repeated families exist in the mouse genome. These are of particular interest because presumably the...

The mouse major urinary proteins (MUPs) are the products of a multi-gene family of 30-35 genes whose members exhibit diverse tissue specific, developmental, and hormonal controls. Three cDNA clones corresponding to liver MUP mRNAs have been sequenced. Two of the clones (p499, C57BL/6 and p1057, BALB/c) share strong homology whereas a third clone (p199, C57BL/6) has diverged considerably from the others at the nucleic acid (85% homology) and protein (68% homology) levels. The 5' regions of p499 and p199 which show the most sequence divergence were subcloned and shown to hybridize to different liver MUP mRNAs. The p499-5' sequence was expressed in all MUP expressing tissues (liver, lachrymal, submaxillary and mammary) whereas the p199-5' sequence was expressed primarily in the liver and lachrymal. Analysis of liver RNA from mice in different endocrine states indicates that the p499-5' sequence is strongly regulated by thyroxine administration whereas the p199-5' sequence is not. Both sequences appear to be regulated by growth hormone and testosterone. Southern blot analysis of mouse genomic DNA indicates that there are multiple genes homologous to each sequence.

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.

A short segment of simian virus 40 (SV40) chromatin on the late side of the origin of replication is hypersensitive to nuclease cleavage. The role of DNA sequence information in this nuclease-sensitive feature was examined by constructing deletion mutations in this region. Deletions were introduced into the inserted segment of in(Or)-1411 (a viable, partially duplicated variant of SV40), and nuclease sensitivity of the inserted segment was compared with that of the unaltered sequences in their normal location in the viral genome. Extended deletions (118 to 161 base pairs) essentially abolished nuclease sensitivity within the inserted segment. Shorter deletions (21 to 52 base pairs) at separate locations retained the nuclease-sensitive feature. In some short-deletion mutants nuclease susceptibility was substantially reduced. We conclude that more than one genetic element in this region contributes to the organization of the nuclease-sensitive feature and that the SV40 72-base repeat is not, in itself, sufficient signal for this feature.