We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41–52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 × 10 5 distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices. The mRNA population specifies a cell’s identity and helps to govern its present and future activities. This has made transcriptome analysis a general phenotyping method, with expression microarrays of many kinds in routine use. Here we explore the possibility that transcriptome analysis, transcript discovery and transcript refinement can be done effectively in large and complex mammalian genomes by ultra-high-throughput sequencing. Expression microarrays are currently the most widely used methodology for transcriptome analysis, although some limitations persist. These include hybridization and cross-hybridization artifacts 1–3 , dye-based detection issues and design constraints that preclude or seriously limit the detection of RNA splice patterns and previously unmapped genes. These issues have made it difficult for standard array designs to provide full sequence comprehensiveness (coverage of all possible genes, including unknown ones, in large genomes) or transcriptome comprehensiveness (reliable detection of all RNAs of all prevalence classes, including the least abundant ones that are physiologically relevant). Other

/pdf/mapping-and-quantifying-mammalian-transcriptomes-by-rna-seq-1egh5oivst.pdf

Mapping and quantifying mammalian transcriptomes by RNA-Seq.

Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I

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.

Evolution by gene duplication

Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7 Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients

https://science.sciencemag.org/content/sci/245/4922/1066.full.pdf

Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.

Hundreds of thousands of copies of DNA sequences have been incorporated into the genomes of higher organisms.

/pdf/repeated-sequences-in-dna-4tcffmb3h2.pdf

Repeated Sequences in DNA

Cell differentiation is based almost certainly on the regulation of gene activity, so that for each state of differentiation a certain set of genes is active in transcription and other genes are inactive. The establishment of this concept (1) has depended on evidence
indicating that the cells of an organism generally contain identical genomes (2). Direct support for the idea that regulation of gene activity underlies cell differentiation
comes from evidence that much of the genome in higher cell
types is inactive (3) and that different ribonucleic acids (RNA) are synthesized in different cell types (4).

http://science.sciencemag.org/content/sci/165/3891/349.full.pdf

Gene Regulation for Higher Cells: A Theory

Repetitive DNA occurs widely, if not universally, among higher organisms. A variety of procedures has been developed or adapted to examine its characteristics and a body of concepts and language has grown up to deal with its complexities. This chapter attempts to summarize this body of knowledge and technique. Owing to human frailty and the real problems of the subject, it tends to emphasize the approaches and conceptual position. The chapter describes techniques for the analysis of repeating DNA sequences by reassociation and a method for the evaluation of rate constants. The purity of the DNA's used in reassociation is critical, as the presence of contaminating proteins and metal ions can markedly alter reassociation results and reproducibility. Metal ions can be removed from DNA by passing it over Chelex 100 (Bio-Rad Labs) that has been neutralized and equilibrated with buffer.

Analysis of repeating DNA sequences by reassociation

Recent experimental information on DNA sequence repetition is reviewed, and the significance of both repetitive and non-repetitive sequence considered. Included are a summary of data on the distribution of genome sizes in animals, new experiments on interspecific DNA homology, the distribution of sequence frequencies, and the interspersion of repetitive sequences within the genome. Aspects of the process of evolution are considered at the level of change in the DNA. the process by which novel structure and function could have arisen during evolution are considered speculatively in terms of the authors' gene regulation theory (Britten and Davidson, 1969).

/pdf/repetitive-and-non-repetitive-dna-sequences-and-a-20aqkdac3q.pdf

Repetitive and non-repetitive DNA sequences and a speculation on the origins of evolutionary novelty.

The mutation rates of DNA sequences during evolution can be estimated from interspecies DNA sequence differences by assaying changes that have little or no effect on the phenotype (neutral mutations). Examination of available measurements shows that rates of DNA change of different phylogenetic groups differ by a factor of 5. The slowest rates are observed for higher primates and some bird lineages, while faster rates are seen in rodents, sea urchins, and drosophila. The rate of DNA sequence change has decreased markedly during primate evolution. The contrast in rates of DNA sequence change is probably due to evolutionary variation and selection of biochemical mechanisms such as DNA replication or repair.

https://science.sciencemag.org/content/sci/231/4744/1393.full.pdf

Roy J. Britten

Papers

Repeated Sequences in DNA

Gene Regulation for Higher Cells: A Theory

Analysis of repeating DNA sequences by reassociation

Repetitive and non-repetitive DNA sequences and a speculation on the origins of evolutionary novelty.

Rates of DNA sequence evolution differ between taxonomic groups