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ABSTRACTcDNAs encoding the complement decay-accelerating factor (DAF) were isolated from HeLa and differentiated HL-60 kgt cDNA libraries by screening with a codon preference oligonucleotide corresponding to DAF NH2-terminal amino acids 3-14. The composite cDNA sequence showed a 347-amino acid protein preceded by an NH2-terminal leader peptide sequence. The translated sequence beginning at the DAF NH2 terminus encodes four contiguous %61-amino acid long repetitive units of internal homology. The repetitive regions contain four conserved cysteines, one proline, one glycine, one glycine/alanine, four leucines/isoleucines/valines, one serine, three tyrosines/phenylalanines, and one tryptophan and show striking homology to similar regions previously identified in factor B, C2, C4 binding protein, factor H, COr, factor XM, interleukin 2 receptor, and serum f82-glycoprotein I. The consensus repeats are attached to a 70-amino acid long segment rich in serine and threonine (potential 0-glycosylation sites), which is in turn followed by a stretch of hydrophobic amino acids. RNA blot analysis of HeLa and HL-60 RNA revealed three DAF mRNA species of 3.1, 2.7, and 2.0 kilobases. The results indicate that portions of the DAF gene may have evolved from a DNA element common to the above proteins, that DAF cDNA predicts a COOH-terminal anchoring polypeptide, and that distinct species of DAF message are elaborated in cells.

Stable mutants with reduced capacity to produce capsules were isolated from suspensions of Cryptococcus neoformans after treatment of the wild type with a mutagen. The mutants could be assigned one of two phenotypes, hypocapsular or acapsular. Hypocapsular mutants were immunochemically and physicochemically indistinguishable from the wild type, whereas acapsular mutants lacked a major capsular antigen and a negatively charged exterior. In genetic analysis, the mutant trait segregated as a Mendelian gene (1:1) when random basidiospores from an outcross were studied, and analysis of products of single meiotic events from outcrossed mutants was likewise consistent with meiotic segregation. Two-factor crosses yielded the expected four classes of progeny, with recombinants equal to parentals. We concluded that chromosomal genes are responsible for synthesis of the cryptococcal capsule and that random basidiospore analysis represents a useful technique for genetic analysis in this species.

Decay-accelerating factor (DAF) is one of a family of cell-associated proteins that undergo posttranslational modifications in which glycolipid anchoring structures are substituted for membrane-spanning sequences. The signals that direct the covalent substitution reaction in these proteins are unknown. Human DAF was expressed in Chinese hamster ovary (CHO) cells and murine BW lymphocytes. In both cases, the xenogeneic DAF in transfectants incorporated a glycolipid anchor. A chimeric CD8-DAF cDNA, encompassing the extracellular region of the T-lymphocyte surface antigen CD8 and the 3' end of DAF mRNA (encoding the C-terminal region of mature DAF as well as the hydrophobic extension peptide), was expressed in human leukemia lines after transfection with an Epstein-Barr virus-based episomal vector. The chimeric protein in transfectants demonstrated glycolipid anchoring, whereas unaltered CD8 in control experiments did not. The signals directing glycolipid anchoring in eukaryotic cells are thus evolutionarily conserved and contained in the 3' end of the DAF sequence.

The bacteriophage P2 ogr gene product is a positive regulator of transcription from P2 late promoters. The ogr gene was originally defined by compensatory mutations that overcame the block to P2 growth imposed by a host mutation, rpoA109, in the gene encoding the a subunit of RNA polymerase. DNA sequence analysis has confirmed that this mutation affects the C-terminal region of the a subunit, changing a leucine residue at position 290 to a histidine (rpoAL290H). We have employed a reporter plasmid system to screen other, previously described, rpoA mutants for effects on activation of a P2 late promoter and have identified a second allele, rpoA155, that blocks P2 late transcription. This mutation lies just upstream of rpoAL290H, changing the leucine residue at position 289 to a phenylalanine (rpoAL289F). The effect of the rpoAL289F mutation is not suppressed by the rpoAL29OH-compensatory P2 ogr mutation. P2 ogr mutants that overcome the block imposed by rpoAL289F were isolated and characterized. Our results are consistent with a direct interaction between Ogr and the a subunit of RNA polymerase and support a model in which transcription factor contact sites within the C terminus of a are discrete and tightly clustered.Many eubacterial genes and operons are under positive control, and the structures and DNA-binding sites of a number of prokaryotic transcriptional activators have been well characterized (for a review, see reference 1). The precise mechanism by which these regulatory proteins catalyze the initiation of transcription remains largely obscure. The identification of lambda repressor (9, 24, 27, 28) and catabolite gene activator protein (CAP) (3, 15, 65) mutants that were defective as activators but retained DNA-binding ability suggested that these proteins function by interacting directly with RNA polymerase. A rapidly growing body of genetic evidence supports a functional interaction between the C-terminal region of the ax subunit(s) of DNA-dependent RNA polymerase and specific transcriptional activators.The earliest indication that the a subunit plays a role in positive control was provided by Escherichia coli mutation rpoA109 (61), which prevents lytic growth of phage P2 and satellite phage P4 by interfering with phage late-gene expression. This mutation results in a leucine-to-histidine change in the C-terminal region of the ax subunit (16 general pattern that has emerged from the characterization of these mutants is that they are altered in the C-terminal region of the a polypeptide and that each rpoA mutation appears to be fairly specific, affecting only one or a small group of positively regulated promoters. Those mutations that affect the responsiveness of RNA polymerase to each activator tend to be tightly clustered, suggesting that each transcriptional activator interacts with a discrete domain within the C-terminal region of the a subunit to stimulate transcription.To further define a domain of the ao subunit involved in interaction with the activators of P2 late-gene expression, we have dete...

Auxotrophic mutants of Cryptococcus neofornans have been obtained by using the methods of mutagenesis and replica-plating. Cryptococcus neoformans is the etiological