Isolation of rapidly evolving genomic sequences: construction of a differential library and identification of a human DNA fragment that does not hybridize to chimpanzee DNA.

A differential library enriched in rapidly evolving human genomic sequences was obtained by phenol-enhanced hybridization of human genomic DNA with an excess of chimpanzee DNA. A DNA fragment 110 bp in length that did not hybridize to either chimpanzee or other primate DNA was identified in this library. It was shown to be a substantially diverged member of the human beta satellite family of tandem repeats. The genomic sequences homologous to the fragment were located on the short arms of human acrocentric chromosomes by in situ hybridization. The human-specific fragment failed to hybridize with RNA from different human tissues. The human-specific fragment exhibits a remarkable level of DNA polymorphism in humans and may be used in the identification of human tissue samples, in the selection of human/rodent somatic cell hybrids containing human acrocentric chromosomes, and in the mapping of these chromosomes.

[Molecular genetic study of the cut locus of Drosophila melanogaster. V. The suffix sequence found in the locus is involved in the 3'-end maturation of different Drosophila mRNAs]. / Molekuliarno-geneticheskoe izuchenie lokusa cut Drosophila melanogaster. Soobshchenie V. Suffiks-posledovatel'nost', obnaruzhennaia v lokuse, vovlechena v 3'-kontsevoe sozrevanie raznykh mRNK drozofily.

The nucleotide sequences of 8 genomic and 2 mRNA copies of the suffix were studied. It was found that this short repeat (265 bp) forms the last exon (73 bp) in different developmentally regulated Drosophila genes. The functioning genes contain short insertions carrying polyadenylation signals and polyadenylation sites at the same position of the suffix. It was shown that the suffix sequence is directly involved in the formation of the last splicing site and 3'-end maturation of mRNA.

[Selection and some properties of recombinant clones of lambda bacteriophage containing structural genes of Drosophila melanogaster]. / Selektsiia i nekotorye svoistva rekombinantnykh klonov faga lambda, soderzhashchikh geny Drosophila melanogaster.

The lambdagt clones containing fragments of D. melanogaster genome were prepared and characterized by hybridization of their DNA with complementary RNA synthesized on the lambdagt DNA, C fragment of lambdagt DNA and total D. melanogaster DNA, and also with mRNA and a stable cytoplasmic poly(A)-RNA both isolated from D. melanogaster cells grown in culture. The technique for a simple selection of hybrid clones is described. The hybridization with mRNA allows one to select the clones containing structural genes of D. melanogaster. It was found that in all cases when the clone contains the structural gene it also contains the reiterated base sequences of the D. melanogaster genome. Several clones containing D. melanogaster DNA fragments with a size of (2--4) X 10(6) daltons which hybridized with a relatively large portion of mRNA were selcted for further analysis.

Replication of chromosomal DNA in diploid Drosophila melanogaster cells cultured in vitro.

Replication rate and replicon sizes in chromosomal DNA of in vitro cultured diploid D. melanogaster cells were determined using autoradiography of 3H-thymidine labeled DNA. Synthesis of DNA in euchromatic and heterochromatic regions of Drosophila diploid cells occurs at different periods of the S phase which last 10 h. During the first 4 h the synthesis is observed only in euchromatic regions. The heterochromatic synthesis starts shortly before the synthesis in euchromatic regions is completed and lasts for 6 h until the end of the S phase. The cells were synchronized by 5-fluorodeoxyuridine which blocked the diploid cell DNA synthesis. Synthesis was found to start simultaneously in most euchromatic replicons. In the majority of the replicons the synthesis started at a single point and proceeded bidirectionally. The average rate of DNA synthesis per fork was 12.5 mum/h (38kb). The mean distance between the middle points of adjacent labeled regions was 70 mgm (210 kb). The size of most replicons ranged from 40 to 120 mum. - These estimates do not apply to the heterochromatic portions of the D. melanogaster genome since the measurements have been carried out on DNA preparations obtained during the first 2 h of the S phase. - On the average, a replicon can consist of 7 chromomeres since the size of a replicon in diploid cell chromosomal DNA and DNA length of a polytene chromomere average 210 and 30 kb, respectively.

[Rate of DNA synthesis and the size of replication units in cell culture of Drosophila melanogaster]. / Opredelenie razmerov replikonov i skorosti replikatsii DNK v kletok Drosophila Melanogaster.

The rate of DNA replication and the size of replication units was examined by means of pulse-labeling with 3H-thymidine and DNA autoradiography in the chromsomes of Drosophila melanogaster synchronized cells culture in vitro. The center-to-center distance between adjacent labeled section of DNA fibers was taken for a size of replication unit, i.e. replicon. DNA synthesis was revealed to start simultaneously in most replicons. The rate of elongation of labeled sections of DNA fibers is about 25 mu per hour (75 kb per hour). Taking into consideration bidirectinal DNA replication the rate of DNA replication per growing point is 12.5 mu per hour (38 kb per hour). The mean size of replicon is about 60 mu (130 kb) in cell culture of Drosophila melanogaster and most of these replicons vary from 20 to 80 mu. Calculation suggests that one replicon may consist of six chromomeres each being known to be formed by a segment of DNA molecular 10 mu long on average.

[Chromosome variability in established cultures of Drosophila melanogaster embryonal cells]. / Khromosomnaia izmenchivost'v peresevaemykh kul'turakh émbrional'nykh kletok Drosophila melanogaster

Diploid cell lines with female karyotype for 7 years conserved the normal chromosome set. Tripoid and tetraploid chromosome sets also remained unchanged after two years of cultivation in both sublines. The karyotypes characterized by supernumerary X-chromosomes or deficient for one or more X-chromosomes are predominant among the aneuploids in diploid and euploid lines. The cloning technique allows to isolate the hyperploid variants only. The following karotypic changes were observed during prolonged cultivation: a loss of a single chromosome from the 4th pair; the X-autosome (third pair) translocation; the enlargement of a heterochromatic pricentromeric segment of the X-chromosome; the centric fusion of the X-chromosomes. The formation of 2 telocentric fragments as a result of the centromeric breakage of the autosome of the second pair is a typical chromosome aberration observed in diploid, triploid and tetraploid cell lines.