Ultrastructural localization of zinc in rat exocrine pancreas by autoradiography

Summary ⁶⁵Zn was infused at a constant rate for 10 days into a rat. Glutaraldehyde fixed, Epon-araldite embedded ultrathin sections of pancreatic tissue were coated with Ilford L4 emulsion and at 211 days exposure were developed. Silver grains were found over the zymogen granules and over the rough endoplasmic reticulum of exocrine cells. Islet tissue was not observed in these studies. The failure of other zinc localization methods to demonstrate zinc in acinar tissue is discussed as are some of the pitfalls of the autoradiographic method and suggestions for future improvement.Published with the approval of the Director of the Wisconsin Agricultural Experiment Station, Madison. Supported in part by USPHS Research Grant AM-05606 from the Nat. Institute of Arthritis and Metabolic Diseases.Supported by an NIH post-doctoral fellowship.     

Induction of recombination between homologous and diverged DNAs by double-strand gaps and breaks and role of mismatch repair.

Sequence homology is expected to influence recombination. To further understand mechanisms of recombination and the impact of reduced homology, we examined recombination during transformation between plasmid-borne DNA flanking a double-strand break (DSB) or gap and its chromosomal homolog. Previous reports have concentrated on spontaneous recombination or initiation by undefined lesions. Sequence divergence of approximately 16% reduced transformation frequencies by at least 10-fold. Gene conversion patterns associated with double-strand gap repair of episomal plasmids or with plasmid integration were analyzed by restriction endonuclease mapping and DNA sequencing. For episomal plasmids carrying homeologous DNA, at least one input end was always preserved beyond 10 bp, whereas for plasmids carrying homologous DNA, both input ends were converted beyond 80 bp in 60% of the transformants. The system allowed the recovery of transformants carrying mixtures of recombinant molecules that might arise if heteroduplex DNA--a presumed recombination intermediate--escapes mismatch repair. Gene conversion involving homologous DNAs frequently involved DNA mismatch repair, directed to a broken strand. A mutation in the PMS1 mismatch repair gene significantly increased the fraction of transformants carrying a mixture of plasmids for homologous DNAs, indicating that PMS1 can participate in DSB-initiated recombination. Since nearly all transformants involving homeologous DNAs carried a single recombinant plasmid in both Pms+ and Pms- strains, stable heteroduplex DNA appears less likely than for homologous DNAs. Regardless of homology, gene conversion does not appear to occur by nucleolytic expansion of a DSB to a gap prior to recombination. The results with homeologous DNAs are consistent with a recombinational repair model that we propose does not require the formation of stable heteroduplex DNA but instead involves other homology-dependent interactions that allow recombination-dependent DNA synthesis.     

Host range temperature-sensitive mutants of herpes simplex virus type 2.

Two small-plaque mutants of herpes simplex virus type 2 (HSV-2) (strain 333), whose growth at 39 C was blocked in certain cell types (cell-dependent temperature sensitivity), were compared compared with parental virus in a number of biological assays. One mutant (no. 69) was found to produce a large number of morphologically normal, but noninfectious, particles; under nonpermissive conditions, these mutant particles were able to interfere with the replication of wild-type HSV-2. The other mutant (no. 74), which is known to belong to a different complementation group, appeared to direct little virus DNA synthesis, even at the permissive temperature. Progeny production and virus DNA synthesis in cells infected by mutant 74 were delayed in comparison with wild-type virus-infected cells. Both mutants were found to be more sensitive to UV irradiation than the parental virus; this was especially marked in the case of mutant 74. Moreover, this mutant was found to have a high transforming efficiency at much lower doses of irradiation than those needed to abolish the cytopathic effect of wildtype HSV-2.     

The development by cytomegalovirus-infected cells of binding affinity for normal human immunoglobulin.

After infection with human cytomegalovirus (CMV), cells develop an affinity for normal human immunoglobulin G (IgG). This was demonstrated using 125iodine-labeled purified IgG. It was further demonstrated that the immunoglobulin molecule binds to CMV-infected cells via its Fc portion, and competition for binding to infected cells occurred between purified preparations of human IgG and the Fc fragment of human IgG. Whole sera from individuals with or without a high titer of anti-CMV antibody were labeled with 125iodine and it was demonstrated that serum from individuals with no anti-CMV antibody had an affinity for CMV-infected cells which probably reflected binding of IgG via its Fc fragment. The possible significance of these results in immunologic studies of human CMV is considered.