Complete mutagenesis of the HIV-1 protease.

Retroviruses encode a protease which needs to be active for the production of infectious virions. A disabling mutation in the protease results in the production of non-infectious virus particles and examination of proteins from these mutant virions reveals unprocessed Gag and Gag-Pol precursor proteins, the substrates of the viral protease. Each amino acid of the HIV-1 protease was individually mutated using a simple mutagenesis procedure which is capable of introducing and identifying missense mutations in each residue of a protein. Phenotypic screening of these mutants in a heterologous assay system reveals three regions within the protease where multiple consecutive amino-acid residues are sensitive to mutation. These results show that random mutagenesis can be used to identify functionally important regions within a protein. Mutants with conditional phenotypes have also been identified within this collection.

Characterization of the gene urf13-T and an unidentified reading frame, ORF 25, in maize and tobacco mitochondria.

We have previously identified two large open reading frames, designated ORF13 and ORF25, in the Texas male-sterile cytoplasm (cms-T) of maize mitochondrial DNA (mtDNA). ORF13 is a single copy gene of chimeric origin that is uniquely transcribed and translated in the mitochondria of cms-T maize, where it produces a polypeptide of approximately 13,000 Mr. The ORF13 reading frame does not occur in the maize N, C or S cytoplasms or Nicotiana tabacum. ORF25 exists as a single copy and is transcribed in the four major maize cytoplasms (N, T, C and S) and N. tabacum. The predicted ORF25 polypeptide has a molecular weight of 24,374 in normal maize and 22,439 in tobacco. Several nucleotide and predicted amino acid changes have occurred in the ORF25 gene among the four maize cytoplasms and N. tabacum. Properties such as transcription and conservation of the sequence between two diverse species suggests that ORF25 encodes a functional plant mitochondrial gene. The ORF25 sequence of maize contains a chloroplast DNA insert homologous to a tRNA-Arg gene; this chloroplast DNA insert is absent in the tobacco ORF25 sequence. Comparison of the ORF25 and ORF13 sequences in restored and non-restored cms-T indicates no differences in their nucleotide sequences. Thus fertility restoration does not alter the primary sequences of ORF13 or ORF25.