Genomic organization of major sperm protein genes and pseudogenes in the nematode Caenorhabditis elegans.

The major sperm proteins (MSPs) are a family of closely related, small, basic proteins comprising 15% of the protein in Caenorhabditis elegans sperm. They are encoded by a multigene family of more than 50 genes, including many pseudogenes. MSP gene transcription occurs only in late primary spermatocytes. In order to study the genomic organization of transcribed MSP genes, probes specific for the 3' untranslated regions of sequenced cDNA clones were used to isolate transcribed genes from genomic libraries. These and other clones of MSP genes were located in overlapping cosmid clones by DNA fingerprinting. These cosmids were aligned with the genetic map by overlap with known genes or in-situ hybridization to chromosomes. Of 40 MSP genes identified, 37, including all those known to be transcribed, are organized into six clusters composed of 3 to 13 genes each. Within each cluster, MSP genes are not in tandem but are separated by at least several thousand bases of DNA. Pseudogenes are interspersed among functional genes. Genes with similar 3' untranslated sequences are in the same cluster. The six MSP clusters are confined to only three chromosomal loci; one on the left arm of chromosome II and two near the middle of chromosome IV. Additional sperm-specific genes are located in one cluster of MSP genes on chromosome IV. The multiplicity of MSP genes appears to be a mechanism for enhancing MSP synthesis in spermatocytes, and the loose clustering of genes could be a result of the mechanism of gene duplication or could play a role in regulation.

Lorist2, a cosmid with transcriptional terminators insulating vector genes from interference by promoters within the insert: effect on DNA yield and cloned insert frequency.

Transcription terminators have been included in a phage-lambda-replicon-based cosmid vector, Lorist2, to insulate vector genes against transcriptional interference from cloned insert DNA. DNA yields of recombinant clones containing Escherichia coli genomic DNA inserts are more even for Lorist2 than with its progenitor LoristB. However, the terminators provide only a partial reduction in the over-representation of r X DNA-containing clones generally observed in cosmid libraries of Caenorhabditis elegans DNA, suggesting that causes other than transcriptional readthrough into the vector contribute to this problem.

DNA sequencing with chain-terminating inhibitors.

A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.

The rDNA of C. elegans: sequence and structure.

We have sequenced one complete rDNA tandem repeat from the nematode C. elegans. By comparative analysis we derive secondary structures for the 18s, 5.8s, and 26s rRNA molecules, and comment on other important features of the sequence. We also present the sequence of a junction between the rDNA and non-ribosomal DNA. Finally, we use our data to quantify the evolutionary relationships among several organisms currently studied in developmental biology.

Use of DNA polymerase I primed by a synthetic oligonucleotide to determine a nucleotide sequence in phage fl DNA.

A sequence of 50 residues in f1 DNA has been determined by the extension of a chemically synthesized octadeoxyribonucleotide by Escherichia coli DNA polymerase I, with radioactive nucleoside triphosphates and f1 DNA template. The polymerized product was synthesized either in the presence of manganese and a mixture of ribo- and deoxyribotriphosphates or in a magnesium-containing reaction with one or more of the four triphosphates absent. The sequence determination depended largely on fractionation of the polymerized products by two-dimensional "homochromatography." This approach and the techniques for the subsequent sequence analysis should be of general use for determining other sequences of DNA. Several features of this sequence suggest that it is located in an intercistronic region of f1 DNA.

Nucleotide sequences of two fragments from the coat-protein cistron of bacteriophage R17 ribonucleic acid.

Bacteriophage R17 RNA was labelled with (32)P and was subjected to partial digestion with ribonuclease T(1). The products were fractionated by ionophoresis on polyacrylamide gel. Two fragments were purified and their nucleotide sequences determined by methods involving complete and further partial digestion with ribonucleases A and T(1). Fragment 20 had a sequence that coded for the amino acids in positions 32-53 of the coat protein of the bacteriophage. Fragment 20X, on further purification in 7m-urea, gave rise to two smaller nucleotides whose sequences coded for the amino acids in positions 56-66 and 67-76 of the coat protein. The sequence of the two fragments was such that they could be written in the form of loops stabilized by base-pairing.

Nucleotide sequence of bacteriophage phi X174 DNA.

A DNA sequence for the genome of bacteriophage phi X174 of approximately 5,375 nucleotides has been determined using the rapid and simple 'plus and minus' method. The sequence identifies many of the features responsible for the production of the proteins of the nine known genes of the organism, including initiation and termination sites for the proteins and RNAs. Two pairs of genes are coded by the same region of DNA using different reading frames.

Sequence and organization of the human mitochondrial genome.

The complete sequence of the 16,569-base pair human mitochondrial genome is presented. The genes for the 12S and 16S rRNAs, 22 tRNAs, cytochrome c oxidase subunits I, II and III, ATPase subunit 6, cytochrome b and eight other predicted protein coding genes have been located. The sequence shows extreme economy in that the genes have none or only a few noncoding bases between them, and in many cases the termination codons are not coded in the DNA but are created post-transcriptionally by polyadenylation of the mRNAs.