Jonathan L. Longmire

Isolation and molecular characterization of a highly polymorphic centromeric tandem repeat in the family falconidae

An abundant tandem repeat has been cloned from genomic DNA of the merlin (Falco columbarius). The cloned sequence is 174 bp in length, and maps by in situ hybridization to the centromeric regions of several of the large chromosomes within the merlin karyotype. Complementary sequences have been identified within a variety of falcon species; these sequences are either absent or in very low copy number in the family Accipitridae. The cloned merlin repeat reveals highly polymorphic restriction patterns in the peregrine falcon (Falco peregrinus). These polymorphisms, which have been shown to be stably inherited within a family of captive peregrines, can be used to differentiate the Greenland and Argentina populations of this endangered raptor species.

Human chromosome-specific repetitive DNA sequences: novel markers for genetic analysis

Two recombinant DNA clones that are localized to single human chromosomes were isolated from a human repetitive DNA library. Clone pHuR 98, a variant satellite 3 sequence, specifically hybridizes to chromosome position 9qh. Clone pHuR 195, a variant satellite 2 sequence, specifically hybridizes to chromosome position 16qh. These locations were determined by fluorescent in situ hybridization to metaphase chromosomes, and confirmed by DNA hybridizations to human chromosomes sorted by flow cytometry. Pulsed field gel electrophoresis analysis indicated that both sequences exist in the genome as large DNA blocks. In situ hybridization to intact interphase nuclei showed a well-defined, localized organization for both DNA sequences. The ability to tag specific human autosomal chromosomes, both at metaphase and in interphase nuclei, allows novel molecular cytogenetic analyses in numerous basic research and clinical studies.

Construction and characterization of partial digest DNA libraries made from flow-sorted human chromosome 16☆

In this report, we present the techniques used for the construction of chromosome-specific partial digest libraries from flow-sorted chromosomes and the characterization of two such libraries from human chromosome 16. These libraries were constructed to provide materials for use in the development of a high-resolution physical map of human chromosome 16, and as part of a distributive effort on the National Laboratory Gene Library Project. Libraries with 20-fold coverage were made in Charon-40 (LA16NL03) and in sCos-1 (LA16NC02) after chromosome 16 was sorted from a mouse-human monochromosomal hybrid cell line containing a single homologue of human chromosome 16. Both libraries are approximately 90% enriched for human chromosome 16, have low nonrecombinant backgrounds, and are highly representative for human chromosome-16 sequences. The cosmid library in particular has provided a valuable resource for the isolation of coding sequences, and in the ongoing development of a physical map of human chromosome 16.

Human release factor eRF1: structural organisation of the unique functional gene on chromosome 5 and of the three processed pseudogenes

In lower and higher eukaryotes, a family of tightly related proteins designated eRF1 (for eukaryotic release factor 1) catalyses termination of protein synthesis at all three stop codons. The human genome contains four eRF1 homologous sequences localised on chromosomes 5, 6, 7 and X. We report here the cloning and the structural analysis of the human eRF1 gene family. It appears that the gene located on chromosome 5 alone is potentially functional, whereas the other three sequences resemble processed pseudogenes. This is the first description of the structural organisation of the human eRF1 gene, which has been remarkably conserved during evolution and which is essential in the translation termination process.

Oncogenes and linkage groups: Conservation during mammalian chromosome evolution

Proto-oncogenes, which represent the cellular progenitors of the transforming genes harbored by acute transforming oncogenic retroviruses, have been highly conserved during vertebrate evolution. In this report, we have assigned experimentally a subset of proto-oncogenes (SRC, ABL, FES, and FMS — all related to the SRC family) to Chinese hamster chromosomes by Southern filter hybridization analyses of DNAs isolated from both somatic cell hybrids and flow-sorted hamster chromosomes. These results demonstrate that several autosomal linkage groups containing proto-oncogenes originated prior to the radiation and speciation of mammals and have remained remarkably stable for nearly 80 million years.

Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosome 3: evidence for the role of chromosome rearrangement in gene amplification.

Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines.

Construction of a cosmid library of DNA replicated early in the S phase of normal human fibroblasts.

We constructed a subgenomic cosmid library of DNA replicated early in the S phase of normal human diploid fibroblasts. Cells were synchronized by release from confluence arrest and incubation in the presence of aphidicolin. Bromodeoxyuridine (BrdUrd) was added to aphidicolin‐containing medium to label DNA replicated as cells entered S phase. Nuclear DNA was partially digested with Sau 3AI, and hybrid density DNA was separated in CsCl gradients. The purified early‐replicating DNA was cloned into sCos1 cosmid vector. Clones were transferred individually into the wells of 96 microtiter plates (9,216 potential clones). Vigorous bacterial growth was detected in 8,742 of those wells. High‐density colony hybridization filters (1,536 clones/filter) were prepared from a set of replicas of the original plates. Bacteria remaining in the wells of replica plates were combined, mixed with freezing medium, and stored at −80°C. These pooled stocks were analyzed by polymerase chain reaction to determine the presence of specific sequences in the library. Hybridization of high‐density filters was used to identify the clones of interest, which were retrieved from the frozen cultures in the 96‐well plates. In testing the library for the presence of 14 known early‐replicating genes, we found sequences at or near 5 of them: APRT, β‐actin, β‐tubulin, c‐myc, and HPRT. This library is a valuable resource for the isolation and analysis of certain DNA sequences replicated at the beginning of S phase, including potential origins of bidirectional replication. J. Cell. Biochem. 78:509–517, 2000.

Chapter 19 Large-Scale Chromosome Sorting

Publisher Summary This chapter focuses on large-scale chromosome sorting for constructing large insert chromosome-specific libraries using cloning vectors such as Charon 40, cosmids, and yeast artificial chromosomes (YACs). Up to 2 μg of sorted chromosomal DNA is commonly used for constructing YAC libraries. This requires the accumulation of 4 to 20 million chromosomes. Requirements for production sorting differ from and are more demanding than the requirements of analytical sorting that typically requires only a few minutes of actual sorting time. For successful production sorting, additional precautions are needed to assure high purity, uninterrupted sorting, and recovery of high-molecular-weight DNA. Chromosome sorting is typically accomplished using chromosomes suspended in a stabilizing buffer and stained with two DNA binding fluorochromes; Hoechst 33258 and chromomycin A3. The binding specificity of each fluorochrome allows one to measure the ratio of adenine-thymine base pairs (A:T) to guanine-cytosine base pairs (G:C) and thereby resolve many more chromosomes in complex karyotypes such as human or mouse than is possible with single fluorochrome staining.

Trans-acting factors in chromosomal instability.

The hypothesis that trans-acting factors affect chromosome stability was explored using human X Chinese hamster somatic cell hybrids. Two types of hybrids were examined. In either case, the human parent consisted of human diploid fibroblasts, the chromosomes of which tended to be lost from the hybrid cell. Comparisons were made between hybrid clones in which the hamster parent had a very stable karyotype (line CHO) and clones from a hamster parent with an unusual ongoing unstable karyotype (line CHX). Chinese hamster-human hybrid cell clones were expanded, and metaphase spreads were analyzed with an in situ hybridization procedure that uses biotin-labeled human genomic DNA as probe. Analyses of chromosome numbers and interspecies translocations were made after 20, 60, and 100 population doublings. Throughout the experiments, the generation of human-hamster-translocated chromosomes was more frequent in the hybrid cells with the CHX background. In addition, these cells also generated human acentric fragments, which were rare in cells with the CHO background. These results favor explanations for the instability of the CHX line that involve ongoing production of a diffusible clastogenic factor.

Use of Variable Number of Tandem Repeat (VNTR) Sequences for Monitoring Chromosomal Instability

Publisher Summary This chapter discusses the monitoring of chromosomal instability by using variable number of tandem repeat (VNTR) sequences. Highly dispersed VNTR families may be useful in assaying the genetic stability of cellular lineages, especially neoplastic ones. The possibility that these sequences are themselves involved in rearrangements that drive the neoplastic process should be considered. Such a possibility could explain the persistent generation of translocations, rearrangements, and deletions commonly seen in solid tumors. Some cytogenetic changes that appear to represent nondisjunctive changes in the number of copies of normal chromosomes are difficult to envision in this way. The data present in the chapter also suggest that fingerprinting may offer the detection of gross chromosomal changes that are not obvious on conventional cytogenetic inspection. Loss of heterozygosity could be extremely important in the neoplastic process, as it could result in the expression of recessive genes such as mutated tumor suppressor genes. The use of VNTR probes would appear to be an ideal way to follow such phenomena, especially if large chromosomal pieces or whole chromosomes are involved.