Biomedical subjects
Publications and source records attributed to M F Singer.
Southern blot analysis of DNA from human-rodent cell hybrids and in situ hybridization to metaphase chromosomes allowed the assignment of a unique human DNA sequence associated with satellite DNA to band p16 of human chromosome 4.
The structure of LINE-1 (L1Ca) family members present on African green monkey chromosome CAE-19 is compared with that of the entire set of L1Ca sequences present in the monkey genome. The analysis involved annealing of cloned subsegments of monkey L1 family members to DNA-blots containing restriction endonuclease digests of either total monkey liver DNA or DNA isolated from a monkey/mouse somatic cell hybrid carrying the single monkey chromosome. In addition, L1Ca segments cloned from hybrid cell DNA were characterized by restriction endonuclease mapping and hybridization. The data indicate that, taken as a whole, the set of L1Ca sequences on CAE-19 tends to differ in characteristic ways from the set present in the total monkey genome.
To investigate the role of satellite DNA in eukaryotic genomes, we isolated from an African green monkey (Cercopithecus aethiops) genomic library cloned segments containing the previously described deca-satellite linked to low-copy-number genomic sequences. Three such clones were obtained. The low-copy-number sequences in the three clones do not cross-hybridize suggesting that they derive from different genomic loci. The structure of one of the clones, lambda MkA, is described in detail. Subcloned segments containing the low-copy-number sequences from lambda MkA anneal to monkey, human and mouse genomic DNA. The subcloned probes were used to select clones containing homologous sequences from a second, independent monkey library as well as from human and mouse genomic libraries. Several of the newly isolated monkey clones hybridized to probes containing the species-specific deca- and alpha-satellites, confirming the genomic association of the low-copy-number sequence in lambda MkA with satellite DNA. Moreover, several of the human and mouse clones hybridized to species-specific human and mouse satellite DNAs, respectively. These experiments indicate that the low-copy-number sequence in lambda MkA and its association with satellite DNA is conserved in primates and rodents.
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The major primate family of highly repeated, long interspersed DNA sequences (LINE-1, previously Kpn I, family) includes several thousand 6-kilobase-pair long units that terminate in an A-rich stretch. Recent evidence indicates that long open reading frames occur in at least some family members. These results suggested that one or more LINE-1 family members might be structural genes. Accordingly, a variety of human cell lines was analyzed for the presence of a cytoplasmic, polyadenylylated RNA homologous to LINE-1 sequences. Such a transcript was detected in a human pluripotent teratocarcinoma cell line (NTera2 clone D1). The RNA is approximately 6.5 kilobases long and is homologous to the LINE-1 strand with the open reading frames. The abundance of the transcript varies markedly with previously described variations in the phenotype of these cells and is highest when the cells display the embryonal carcinoma morphology. This RNA may represent a mRNA transcribed from one or more functional genes in the LINE-1 family.
Previously we reported the existence of a highly polymorphic satellite, deca-satellite, in the African green monkey genome; deca-satellite probe anneals to complex sets of repeated restriction endonuclease fragments that differ from individual to individual in the monkey population. Here we present experiments aimed at clarifying the structure and organization of deca-satellite sequences and investigating the mechanisms that generate the polymorphisms. Deca-satellite represents less than 1% of the monkey genome but the percentage varies from one monkey to another. The core sequence 5'-C-C-G-G within the ten base-pair deca-satellite repeat unit is well conserved and the central 5'-C-G is sometimes but not always methylated. Restriction endonuclease analysis with BamHI and EcoRI defines separate satellite domains that have evolved in an independent manner. In situ hybridization shows deca-satellite to be located at the centromeric regions of some but not all monkey chromosomes. This location is independently confirmed by a high frequency, in monkey libraries, of segments containing junctions between deca-satellite and alpha-satellite, the main monkey centromeric satellite. The total number of metaphase chromosomes that show centromeric grains after in situ hybridization with a deca-satellite probe varies from one monkey to another. Moreover, in situ hybridization to endoreduplicated diplochromosomes showed that deca-satellite is occasionally distributed asymmetrically on one or the other of the two pairs of sister chromatids in one diplochromosome. This indicates that major reorganization of the satellite can occur frequently in somatic cells. We discuss several possible mechanisms by which deca-satellite sequences could be either amplified or deleted during a single replicative cycle. Also, on the basis of the marked fluidity of deca-satellite abundance and organization and other well-known attributes of centromeric satellites, we suggest that the existence and maintenance of centromeric satellite rests on the role of the tandem repeats themselves and not on any particular nucleotide sequence, repeat length or organization.
The ability of an African green monkey genomic segment, homologous to the regulatory region of SV40, to promote transcription in monkey cells has been investigated. Genomic transcripts from CV-1 cells hybridize to both strands of this SV40-like segment. Further examination of the promoter potential of the SV40-like segment using expression vectors suggests that the SV40-like region contains at least part of the necessary information for promoting transcription in both directions. In addition, the latter experiments suggest that sequences several hundred nucleotides away in the genome modulate the transcription initiating at multiple sites in the SV40-like segment.
Comparison of the sequences at the ends of several newly cloned and full length members of the monkey KpnI family with one another and with previously described monkey and human segments defines the nucleotide sequence at the two termini. No terminal repeats either direct or inverted are noted within full length family members which may or may not be immediately flanked by direct repeats. At the 3' terminus, several family members have polyadenylation signals followed by a d(A)-rich stretch. The genomic frequency of segments within the full length element increases markedly from the 5' to the 3' terminus, consistent with the cloning of various truncated family members. One such truncated version joined to a low copy number DNA segment is inserted in monkey alpha-satellite where the combination appears to have been amplified in conjunction with the satellite itself.
Segments of monkey genomic DNA that are homologous to the control region around the origin of replication of simian virus 40 were previously cloned and characterized (Queen et al., Mol. Cell Biol. 1:1061-1068, 1981). We describe here two DNase I-hypersensitive sites that map in the region of monkey chromatin around one such ori-like segment. One of these sites lies within the simian virus 40 homologous segment which is also a site from which transcription initiates bidirectionally (J. Saffer and M. Singer, submitted for publication).
The KpnI and BamH1 (or M1F-1) families are the predominant sets of long interspersed repeated DNA sequences (LINEs) in primates and rodents, respectively. Recently, the sequences of several cloned subsegments from each family were determined in different laboratories. These sequences have now been compared and found to be homologous over at least 1400 bp. The data suggest that the two LINE families had a common progenitor and have been conserved in similar abundance although in divergent forms in the two mammalian orders.
Three different cloned segments of African green monkey DNA that contain alpha-satellite sequences linked to a previously undescribed, distinct monkey satellite (called deca-satellite) are described here. The cloned segments were derived from a monkey DNA library in lambda Charon 4A that was constructed to select for junctions between alpha-satellite and other DNA sequences. The structure of the deca-satellite and of a junction between deca-satellite and alpha-satellite were studied by subcloning appropriate fragments of the original cloned segments and by sequence analysis. Deca-satellite has a ten base-pair repeat unit; the consensus sequence of the repeat units is 5' A-A-A-C-C-G-G-N-T-C. Sequences homologous to the deca-satellite are in the middle repeated class of genomic DNA. Analysis of the organization of deca-satellite sequences by digestion of total DNA with various restriction endonucleases and hybridization with a cloned deca-satellite probe revealed extensive polymorphism in the genomes of different individual monkeys but not among the tissues of one organism. These observations indicate that the arrangement of deca-satellite sequences is continually changing. An unusual alpha-satellite repeat unit occurs at a junction between the alpha-satellite and deca-satellite. It resembles the major baboon alpha-satellite more closely than it does monkey alpha-satellite and thereby provides evidence in favor of the "library" hypothesis for satellite evolution.
Three different members of a family (KpnI-family) of interspersed repeated DNA sequences were found linked to alpha-satellite sequences in cloned segments of the African green monkey genome. In two of these segments the KpnI-family member is over 6 kbp in length and one of them is flanked by alpha-satellite on both sides indicating that it was inserted into a satellite array. Hybridization of subcloned portions of the family members to restriction endonuclease digests of monkey and human DNA and to a genomic library of African green monkey DNA indicate that 1) family members are interspersed in both the monkey and human genomes, 2) some family members may include sequences in addition to those in the three characterized here, 3) some family members may contain only parts of the sequences characterized here and 4) while the overall organization of the family is similar in the human and monkey genome the majority of the family members in each of the two genomes are distinctly identified by the variant position of certain restriction endonuclease sites. This last observation suggests that within each genome there is a tendency to maintain particular versions of the sequence. Observations 2) and 3) suggest that the KpnI family is complex and includes a variety of subfamilies.
An approximately equal to 2-kilobase-pair-long member (Kpn I-LS1) of the African green monkey Kpn I family of repeated sequences has been cloned, subjected to sequence analysis, and compared to other family members which are over 6 kilobase pairs (Kpn I-alpha 7) and 829 base pairs (Kpn I-RET) long. Both Kpn I-LS1 and Kpn I-RET lack sequences found at the ends of the longer family member and their structures resemble those of processed genes. Kpm I-LS1 sequences are colinear with part of the long family member, Kpn I-alpha 7. However, although all sequences in Kpn I-RET are represented in Kpn I-LS1, the two are not colinear; Kpn I-RET is missing 731 base pairs found in Kpn I-LS1 and one segment flanking the deletion is inverted. The results demonstrate that Kpn I family members are not only of different lengths but may also contain scrambled arrangements of common sequences. Sequences in Kpn I-LS1 hybridize to RNA from monkey and human cells, indicating that some family members are transcribed.
We describe here the interruption of a cloned African green monkey alpha-satellite array by an 829-base-pair-long nonsatellite DNA segment. Hybridization experiments indicate that the sequences within the interruption are homologous to segments frequently found in the 6-kilobase-pair-long members of the KpnI family of long, interspersed repeats. These data confirm and extend earlier results suggesting that sequences common to the KpnI family can occur independently of one another and in segments of variable lengths. The 829-base-pair-long segment, which is termed KpnI-RET, contains a terminal stretch of adenosine residues preceded by two typical but overlapping polyadenylation sites. KpnI-RET is flanked by direct repeats of a 14-base-pair-long segment of alpha-satellite that occurs only once in the satellite consensus sequence. These structural features suggest that KpnI-RET was inserted into the satellite array as a movable element.
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Explore the source record for details and available documents.