No abstract

Drosophila melanogaster mitochondrial DNA (mtDNA) is closely related to the mammalian and amphibian mtDNA except for gene organization. In Drosophila, genes are distributed in clusters alternatively coded on each strand. Besides the eleven major foreseeable transcripts previously described (MERTEN and PARDUE, 1981, J. Mol. Biol., 153, 1-21), we have characterized two poly A+ transcripts, one major and one minor which could correspond respectively to the ND3 and ND6 reading frames, and 27 poly A+ minor transcripts (0.2 to greater than 3.2 kb) which are distributed along the mtDNA except in the rRNAs, ND 1 and A+ T rich regions. The mapping and length of 25 of these transcripts strongly suggest a precursor role. They would be processed at the level of tRNA or tRNA-like sequences. Most of them are transcribed from the template strand of each gene cluster and their distribution is in agreement with the hypothesis of several transcription origins and terminations located near the extremities of each gene cluster. Quantitatively our results show a large variation in each presumptive mature transcript compared to the other, even in a given gene cluster, suggesting a specific degradation of some of the mature transcripts.

Two proteins, PS1 and PS2, were detected in the culture medium of Corynebacterium glutamicum and are the major proteins secreted by this bacterium. No enzymatic activity was identified for either of the two proteins. Immunologically cross-reacting proteins were found in a variety of C. glutamicum strains but not in the coryneform Arthrobacter aureus. The gene encoding PS1, csp1, was cloned in lambda gt11 using polyclonal antibodies raised against PS1 to screen for producing clones. The csp1 gene was expressed in Escherichia coli, presumably from its own promoter, and directed the synthesis of two proteins recognized by anti-PS1 antibodies. The major protein band, of lower M(r), was detected in the periplasmic fraction. It had the same M(r) as the PS1 protein band detected in the supernatant of C. glutamicum cultures and presumably corresponds to the mature form of PS1. The minor protein band appears to be the precursor form of PS1. The nucleotide sequence of the csp1 gene was determined and contained an open reading frame encoding a polypeptide with a calculated molecular weight of 70,874, with a putative signal peptide with a molecular weight of 4411. This is consistent with the M(r) determined for PS1 from C. glutamicum culture supernatant and E. coli whole-cell extracts. The NH2-half of the deduced amino acid is similar (about 33% identical residues and 52% including similar residues) to the secreted antigen 85 protein complex of Mycobacterium. The csp1 gene in C. glutamicum was disrupted without any apparent effect on growth or viability.

Abstract.A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick-translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: (a) rapid signal detection; (b) superior morphological preservation and spatial resolution; and (c) signal-to-noise ratios equivalent to radiolabeling.