Inter-species DNA polymerase delta chimeras are functional in Saccharomyces cerevisiae.

The catalytic subunits of DNA polymerase delta of Schizosaccharomyces pombe and Saccharomyces cerevisiae share over 50% identity. The capability of S. pombe DNA polymerase delta to complement two thermosensitive mutants of S. cerevisiae was studied in vivo and it was determined that complementation was allele dependent. However, DNA polymerase delta from S. pombe did not restore growth of a S. cerevisiae strain containing a disrupted chromosomal copy of the POL3 gene that encodes DNA polymerase delta. To identify the regions of DNA polymerase delta responsible for species-specific interactions, we constructed different chimeras with S. cerevisiae and S. pombe DNA polymerase delta genes. The growth of a S. cerevisiae strain with a disrupted chromosomal POL3 gene was studied after transformation with plasmids expressing different chimeras. A 1254-bp region located in the 3' region of the S. cerevisiae POL3 gene is responsible for species-specific functions.

Expression of the catalytic subunits of pol alpha and pol delta from fission yeast Schizosaccharomyces pombe.

This paper reports on expression and posttranslational modifications of the catalytic subunits of pol alpha and pol delta from fission yeast Schizosaccharomyces pombe. Okadaic acid treatment of S. pombe spheroplasts in amounts known to inhibit phosphatases 1 and 2A resulted in decreased proteolysis of both pol alpha and pol delta. Computer analysis of pol alpha and pol delta sequences confirmed the presence of consensus motifs for protein phosphorylation. Indirect immunofluorescence microscopy of S. pombe cells showed nuclear location of both proteins in wild type cells. However, whereas cells transformed with a vector expressing pol alpha produced a clear increase of the nuclear signal, no increase was detectable in cells transformed with pol delta. This observation suggests the existence of a mechanism limiting the cell concentration of pol delta in the cell. Constitutive expression of S. pombe pol delta in E. coli was possible only with vectors containing truncated forms of its gene, indicating a toxic effect of pol delta on E. coli growth.

Mutations in the D strand of the human CD4 V1 domain affect CD4 interactions with the human immunodeficiency virus envelope glycoprotein gp120 and HLA class II antigens similarly.

CD4, a cell surface glycoprotein expressed primarily by T lymphocytes and monocytes, interacts with HLA class II antigens to regulate the immune response. In AIDS, CD4 is the receptor for the human immunodeficiency virus, which binds to CD4 through envelope glycoprotein gp120. Delineation of the ligand-binding sites of CD4 is necessary for the development of immunomodulators and antiviral agents. Although the gp120 binding site has been characterized in detail, much less is known about the class II binding site, and it is as yet uncertain whether they partially or fully overlap. To investigate CD4 binding sites, a cellular adhesion assay between COS cells transiently transfected with CD4 and B lymphocytes expressing HLA class II antigens has been developed that is strictly dependent on the CD4--class II interaction, quantitative, and highly reproducible. Mutants of CD4 expressing amino acids with distinct physicochemical properties at positions Arg-54, Ala-55, Asp-56, and Ser-57 in V1, the first extracellular immunoglobulin-like domain, have been generated and studied qualitatively and quantitatively for interaction with HLA class II antigens, for membrane expression, for the integrity of CD4 epitopes recognized by a panel of monoclonal antibodies, and for gp120 binding. The results obtained show that the mutations in this tetrapeptide, which forms the core of a synthetic peptide previously shown to have immunosuppressive properties, affect the two binding functions of CD4 similarly, lending support to the hypothesis that the human immunodeficiency virus mimicks HLA class II binding to CD4.