Haematozoa of fishes in Humboldt Bay, California.

Five hundred seven fish representing 45 species from Humboldt Bay, California (USA) were examined for blood parasites. Four fish (less than 1%) from two species were infected. Haemogregarina leptocotti sp. n. is described from one of 33 staghorn sculpin (Leptocottus armatus). Haemogregarina roelofsi sp. n. is described from three of 15 black rockfish (Sebastes melanops). Gametocytes of H. leptocotti sp. n. averaged 6.1 x 2.1 microns with a 2.7 x 1.7 microns oval nucleus; those of H. roelofsi sp. n. averaged 5.5 x 2.7 microns with a 2.5 x 2.2 microns rectangular nucleus. Neither species of parasite had distinct chromatin granules, a polar cap, or more than one gametocyte in an infected cell. Haematozoa are relatively rare in fishes of the northeastern Pacific Ocean.     

Strand breaks without DNA rearrangement in V (D)J recombination.

Somatic gene rearrangement of immunoglobulin and T-cell receptor genes [V(D)J recombination] is mediated by pairs of specific DNA sequence motifs termed signal sequences. In experiments described here, retroviral vectors containing V(D)J rearrangement cassettes in which the signal sequences had been altered were introduced into wild-type and scid (severe combined immune deficiency) pre-B cells and used to define intermediates in the V(D)J recombination pathway. The scid mutation has previously been shown to deleteriously affect the V(D)J recombination process. Cassettes containing a point mutation in one of the two signal sequences inhibited rearrangement in wild-type cells. In contrast, scid cells continued to rearrange these cassettes with the characteristic scid deletional phenotype. Using these mutated templates, we identified junctional modifications at the wild-type signal sequences that had arisen from strand breaks which were not associated with overall V(D)J rearrangements. Neither cell type was able to rearrange constructs which contained only a single, nonmutated, signal sequence. In addition, scid and wild-type cell lines harboring cassettes with mutations in both signal sequences did not undergo rearrangement, suggesting that at least one functional signal sequence was required for all types of V(D)J recombination events. Analysis of these signal sequence mutations has provided insights into intermediates in the V(D)J rearrangement pathway in wild-type and scid pre-B cells.     

Sequence specificity for the initiation of RNA-primed simian virus 40 DNA synthesis in vivo

Analysis of the nucleotide sequences at the 5' ends of RNA-primed nascent DNA chains (Okazaki fragments) and of their locations in replicating simian virus 40 (SV40) DNA revealed the precise nature of Okazaki fragment initiation sites in vivo. The primary initiation site for mammalian DNA primase was 3'-purine-dT-5' in the DNA template and the secondary site was 3'-purine-dC-5', with the 5' end of the RNA primer complementary to either the dT or dC. The third position of the initiation site was variable with a preference for dT or dA. About 81% of the available 3'-purine-dT-5' sites and 20% of the 3'-purine-dC-5' sites were used. Purine-rich sites, such as PuPuPu and PyPuPu , were excluded. The 5'-terminal ribonucleotide composition of Okazaki fragments corroborated these conclusions. Furthermore, the length of individual RNA primers was not unique, but varied in size from six to ten bases with some appearing as short as three bases and some as long as 12 bases, depending on the initiation site used. This result was consistent with the average size (9 to 11 bases) of RNA primers isolated from specific regions of the genome. Excision of RNA primers did not appear to stop at the RNA-DNA junction, but removed a variable number of deoxyribonucleotides from the 5' end of the nascent DNA chain. Finally, only one-fourth of the replication forks contained an Okazaki fragment, and the distribution of their initiation sites between the two arms revealed that Okazaki fragments were initiated exclusively (99%) on retrograde DNA templates. The data obtained at two genomic sites about 350 and 1780 bases from ori were essentially the same as that reported for the ori region (Hay & DePamphilis , 1982), suggesting that the mechanism used to synthesize the first DNA chain at ori is the same as that used to synthesize Okazaki fragments throughout the genome.     

Dedication to Dr. Robert “Bob” Rush Miller

Reviews in Fish Biology and Fisheries -     

A fluorescent substrate for the assay of phosphatidylinositol-specific phospholipase C: 4-(1-pyreno)butylphosphoryl-1-myo-inositol

Racemic 4-(1-pyreno)butylphosphoryl-1-myo-inositol was synthesized from a pentaprotected inositol-1-dimethylphosphite by phosphite coupling with 4-(1-pyreno)butanol. It is a good substrate for a very sensitive assay of phosphatidylinositol-specific phospholipase C.