We describe a new basis for the construction of a genetic linkage map of the human genome. The basic principle of the mapping scheme is to develop, by recombinant DNA techniques, random single-copy DNA probes capable of detecting DNA sequence polymorphisms, when hybridized to restriction digests of an individual's DNA. Each of these probes will define a locus. Loci can be expanded or contracted to include more or less polymorphism by further application of recombinant DNA technology. Suitably polymorphic loci can be tested for linkage relationships in human pedigrees by established methods; and loci can be arranged into linkage groups to form a true genetic map of "DNA marker loci." Pedigrees in which inherited traits are known to be segregating can then be analyzed, making possible the mapping of the gene(s) responsible for the trait with respect to the DNA marker loci, without requiring direct access to a specified gene's DNA. For inherited diseases mapped in this way, linked DNA marker loci can be used predictively for genetic counseling.

Construction of a genetic linkage map in man using restriction fragment length polymorphisms.

It is concluded from a review of the literature that plant cell culture itself generates genetic variability (somaclonal variation). Extensive examples are discussed of such variation in culture subclones and in regenerated plants (somaclones). A number of possible mechanisms for the origin of this phenomenon are considered. It is argued that this variation already is proving to be of significance for plant improvement. In particular the phenomenon may be employed to enhance the exchange required in sexual hybrids for the introgression of desirable alien genes into a crop species. It may also be used to generate variants of a commercial cultivar in high frequency without hybridizing to other genotypes.

Somaclonal variation — a novel source of variability from cell cultures for plant improvement

https://www.cell.com/cell/pdf/0092-8674(84)90301-5.pdf

Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis

Natural selection operating within genomes will inevitably result in the appearance of DNAs with no phenotypic expression whose only ‘function’ is survival within genomes. Prokaryotic transposable elements and eukaryotic middle-repetitive sequences can be seen as such DNAs, and thus no phenotypic or evolutionary function need be assigned to them.

Selfish genes, the phenotype paradigm and genome evolution.

Repetitive DNA sequences form a large portion of the genomes of eukaryotes. The 'selfish DNA' hypothesis proposes that they are maintained by their ability to replicate within the genome. The behaviour of repetitive sequences can result in mutations that cause genetic diseases, and confer significant fitness losses on the organism. Features of the organization of repetitive sequences in eukaryotic genomes, and their distribution in natural populations, reflect the evolutionary forces acting on selfish DNA.

The evolutionary dynamics of repetitive DNA in eukaryotes

In the polymerase chain reaction (PCR), two specific oligonucleotide primers are used to amplify the sequences between them. However, this technique is not suitable for amplifying genes that encode molecules where the 5' portion of the sequences of interest is not known, such as the T cell receptor (TCR) or immunoglobulins. Because of this limitation, a novel technique, anchored polymerase chain reaction (A-PCR), was devised that requires sequence specificity only on the 3' end of the target fragment. It was used to analyze TCR delta chain mRNA's from human peripheral blood gamma delta T cells. Most of these cells had a V delta gene segment not previously described (V delta 3), and the delta chain junctional sequences formed a discrete subpopulation compared with those previously reported.

https://science.sciencemag.org/content/sci/243/4888/217.full.pdf

Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain.

Evidence for transposition of dispersed repetitive DNA families in yeast

The RNA homologous to the yeast transposable element Ty1 is one of the more abundant poly(A)+ RNAs in many strains of the yeast Saccharomyces cerevisiae. The 5' and 3' ends of Ty1 RNA have been determined from analysis of cDNA. The 5' end is 245 bases into the left delta sequence measured from the left side of the Ty1 element. The delta sequence is a direct repeat of about 340 base pairs present at each end of the Ty1 element. The Ty1 transcription includes 93-97 bases of the left delta sequence and continues through the entire internal portion of the element and through about 295 bases of the right delta sequence before reaching the 3' end located 38-46 bases from the right side of the right delta sequence. Because the delta sequences present at each end of a single Ty1 element have identical or very similar DNA sequences, these end points for Ty1 RNA raise several questions about the expression of Ty1 elements. First, what are the initiation and termination signals, because the Ty1 transcript must read through a DNA sequence that is identical to the 3' end at about 50 bases from the 5' end? Second, why is the direction of transcription of the Ty1 element opposite to that of genes that are overexpressed after the insertion of a Ty1 element? Third, because the Ty1 RNA itself has direct repeats of about 45 bases, a structure analogous to retrovirus RNAs, is the Ty1 RNA an intermediate in the transposition of Ty1?

https://www.pnas.org/content/pnas/80/9/2432.full.pdf

RNA from the yeast transposable element Ty1 has both ends in the direct repeats, a structure similar to retrovirus RNA.

Thymus-derived lymphocytes (T cells) use clonally distributed antigen receptors to recognize peptide fragments associated with products of the major histocompatibility complex (MHC) (refs 1–4). On most murine and human T cells the T cell receptor (TCR) is composed of disulphide-linked α and β chains (TCRα/β), each of which contains constant and variable domains, and which are associated with the invariant chains of the CD3 complex5,6. It has been demonstrated, however, that a distinct CD3-associated TCR is expressed on a small subset of T cells or immature thymocytes which fail to express either CD4 or CD8 (refs 7–14), the molecules associated with class II or class I MHC antigen recognition. Instead of TCRα/β, these cells express heterodimers of γ and δ chains (TRCγ/δ). The genes encoding α, β, and γ have been isolated and characterized15–17. A new murine T cell receptor (Cx) gene which undergoes rearrangement and expression early during T cell ontogeny has recently been identified 5′ of the murine JαCα gene locus18. Here we isolate and sequence the homologous transcript from PEER, a human cell line that expresses a TCRγ/δ, and show that it encodes a protein with characteristic V, D, J, and C segments. Using probes derived from this transcript, we have shown that both PEER and MOLT-13, another TCRγ/δ-expressing cell line, rearrange this locus and express two sizes of transcripts differing in the 3' untranslated region. Using a synthetic peptide derived from the deduced C region sequence, we have prepared antisera that precipitates the δ chain of the TCR from both PEER and MOLT-13, thus demonstrating that Cx and its human homologue code for the δ chain of the TCR.

Identification and sequence of a fourth human T cell antigen receptor chain.

The locus of the delta chain of the human T-cell receptor has been isolated and examined. Three D (diversity) regions and two J (joining) regions are present on the 5' side of the C (constant) region. The closest V (variable) region to the constant region is V delta 2, which in the germ line is found on the 3' side of the constant region in an inverted direction. The genomic structure of the human locus closely parallels its mouse counterpart. Several cDNA sequences and a series of rearranged genomic sequences are compared which demonstrate an enormous potential diversity in the junctional region, between the variable region and the joining region. We find the predominant utilization of the PEER variable region in thymic polyclonal gamma delta cell lines and in some peripheral blood gamma delta cell lines. Thus, the delta chain may have relatively limited variable-region diversity but a large junctional-region diversity. The implications of this observation are discussed.

Elwyn Y. Loh

Papers

Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain.

Evidence for transposition of dispersed repetitive DNA families in yeast

RNA from the yeast transposable element Ty1 has both ends in the direct repeats, a structure similar to retrovirus RNA.

Identification and sequence of a fourth human T cell antigen receptor chain.

Human T-cell-receptor delta chain: genomic organization, diversity, and expression in populations of cells