Computer modelling of DNA structures involved in chromosome maintenance.

Sequence-dependent DNA bending of synthetic and natural molecules was studied by computer analysis. Modelling of synthetic oligonucleotides and of 107 kb of natural sequences gave results which closely resembled published electrophoretic data, demonstrating the powerful predictive capacity of the procedure. The analysis was extended to the study of DNA structures involved in chromosome maintenance. Centromeric DNAs from yeast were found to have sequences in their functional elements which cause them to be unusually straight. Autonomous replicating sequences were found to have two structural domains, one consisting of unusually straight sequences surrounding the consensus and the other of bending elements in flanking DNA. In addition to a structural homology, centromeric and autonomous replicating sequences share common sequence elements. These observations show that computer modelling of natural sequences is a viable approach to the study of the biological implications of alternative DNA structures.     

Cell cycle regulated synthesis of stable mouse thymidine kinase mRNA is mediated by a sequence within the cDNA.

The cDNA for mouse thymidine kinase (TK) was isolated from a cDNA library in lambda-gt11 and sequenced. It was used as a probe to follow the time course of TK mRNA expression in growth stimulated mouse fibroblasts. Linked to the HSV-TK promoter the cDNA was able to transform LTK-cells to the TK+ phenotype. The transformed cells expressed the TK mRNA and enzyme activity in a growth dependent fashion suggesting that the regulatory element is localized on the cDNA.     

The synthesis of protected 5''-amino-2'',5''-dideoxyribonucleoside-3''-O-phosphoramidites; applications of 5''-amino-oligodeoxyribonucleotides.

Synthetic routes to the four appropriately protected 5'-amino-2',5'-dideoxyribonucleoside-3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) have been developed. The structures of all intermediates were confirmed by 13C n.m.r. spectroscopy. These building blocks have been used to prepare 5'-amino-oligodeoxyribonucleotides, which can be coupled to a wide variety of compounds, in particular metal cluster derivatives, but also fluorophores and biotin derivatives, thus generating a variety of very useful probes. Brief mention is made of a tetrairidium cluster derivative of 5'-amino-d[CCGATATCGG], which has been cocrystallised with EcoRV, and will be used for electron microscopy studies.     

The chicken carbonic anhydrase II gene: evidence for a recent shift in intron position.

The complete nucleotide sequence of the coding region of the chicken carbonic anhydrase II (CA II) gene has been determined from clones isolated from a chicken genomic library. The sequence of a nearly full length chicken CA II cDNA clone has also been obtained. The gene is approximately 17 kilobase pairs (kb) in size and codes for a protein that is comprised of 259 amino acid residues. The 5' flanking region contains consensus sequences commonly associated with eucaryotic genes transcribed by RNA polymerase II. Six introns ranging in size from 0.3 to 10.2 kb interrupt the gene. The number of introns as well as five of the six intron locations are conserved between the chicken and mouse CA II genes. The site of the fourth intron is shifted by 14 base pairs further 3' in the chicken and thus falls between codons 147 and 148 rather than within codon 143 as in the mouse gene. Measurements of CA II RNA levels in various cell types suggest that CA II RNA increases in parallel with globin RNA during erythropoiesis and exists only at low levels, if at all, in non-erythroid cells.     

Identification of 28 DNA fragments that detect RFLPs in 13 distinct physical regions of the short arm of chromosome 5.

A series of 175 lambda phage carrying human inserts isolated from a library that is specific for the short arm of human chromosome 5 (5p) have been regionally mapped on 5p using a deletion mapping panel of 16 human-hamster cell hybrids, each of which contains a chromosome 5 with a different deletion in the short arm. Seventy-five single copy DNA fragments were screened with 12 restriction enzymes for their ability to detect restriction fragment length polymorphisms (RFLPs). Twenty-eight of these DNA fragments, which are located in 13 distinct physical regions of 5p, were found to detect RFLPs. These DNA markers make it possible to construct a linkage map that will span the entire length of 5p and will allow the relationship between genetic and physical distance for this region of the genome to be examined at a high level of resolution.