Cvt18/Gsa12 is required for cytoplasm-to-vacuole transport, pexophagy, and autophagy in Saccharomyces cerevisiae and Pichia pastoris.

Eukaryotic cells have the ability to degrade proteins and organelles by selective and nonselective modes of micro- and macroautophagy. In addition, there exist both constitutive and regulated forms of autophagy. For example, pexophagy is a selective process for the regulated degradation of peroxisomes by autophagy. Our studies have shown that the differing pathways of autophagy have many molecular events in common. In this article, we have identified a new member in the family of autophagy genes. GSA12 in Pichia pastoris and its Saccharomyces cerevisiae counterpart, CVT18, encode a soluble protein with two WD40 domains. We have shown that these proteins are required for pexophagy and autophagy in P. pastoris and the Cvt pathway, autophagy, and pexophagy in S. cerevisiae. In P. pastoris, Gsa12 appears to be required for an early event in pexophagy. That is, the involution of the vacuole or extension of vacuole arms to engulf the peroxisomes does not occur in the gsa12 mutant. Consistent with its role in vacuole engulfment, we have found that this cytosolic protein is also localized to the vacuole surface. Similarly, Cvt18 displays a subcellular localization that distinguishes it from the characterized proteins required for cytoplasm-to-vacuole delivery pathways.

Molecular cloning and chromatin structure analysis of the murine alpha1(I) collagen gene domain.

We have isolated molecular clones of genomic mouse DNA spanning 55 kb, including the entire coding region of the murine alpha1(I) collagen (Col1a1) gene and 24 kb of 5' and 13 kb of 3'-flanking sequences, and have performed a detailed chromatin structure analysis of these sequences. Several new DNase-I-hypersensitive sites were identified. The distal 5'-flanking region contains two clusters of DNase-I-hypersensitive sites located between 7 and 8 kb and between 15 and 20 kb upstream of the start site of transcription, respectively. Several of these sites were shown to be present in collagen-producing, but not in non-producing cells, indicating that they are associated with transcription of the gene and may function in its regulation. One strong constitutive DNase-I-hypersensitive site at -18.5 kb was also cleaved by endogenous nucleases. The 3'-flanking region of the gene contains a DNase-I-hypersensitive site located 6 kb downstream of the end of the gene, as well as sequences that can induce a non-B DNA structure. Because these latter sequences coincide with DNase-I-hypersensitive sites in the homologous human gene, our results suggest that some regulatory elements may play a role in gene regulation, not by specific protein-DNA interactions but by virtue of their ability to induce a non-B DNA structure and/or an alternate chromatin conformation. A comparison of the murine and human Col1a1 domains shows a similar, although not identical, distribution of DNase-I-hypersensitive sites, indicating a conserved arrangement of regulatory elements. Our results strongly suggest that these new sites constitute regulatory elements which are involved in the transcriptional regulation and/or chromatin loop organization of the Col1a1 gene, and they are now amenable for functional analyses.

Risk Analysis and Its Application in FSIS.

Risk analysis has become a well established field in the United States during the past two decades; it is used very effectively in several application areas to assess and manage risk. Advances in the application of risk analysis to a wider range of hazards, including biological hazards, and scenarios are now occurring. Definitions of risk analysis terms (including risk assessment, risk management, and risk communication) have been developed along with the rationale, purpose, and need for risk analysis in regulation. Generic principles and applications are presented. A risk analysis program for foodborne hazards is being developed by the Food Safety and Inspection Service (FSIS) of the U.S. Department of Agriculture. A food risk analysis program for meat and poultry is being designed and some potential projects for risk assessment have been selected. All types of foodborne hazards (biological chemical, and physical) will be addressed in the new program. A general overview of risk assessment procedures and the organizational structure for the program are presented. National and international food risk analysis activities by the FSIS are summarized. The Agency expects the new risk analysis program to facilitate the regulation of FSIS-inspected food products.

Characterization of cell surface properties in agglutinable and nonagglutinable mutants of Pseudomonas putida.

Cells of an aggressive, root-colonizing isolate of Pseudomonas putida are agglutinated by a root surface glycoprotein. The agglutination phenotype in P. putida isolate Corvallis is lacking in mutants (Agg-) derived by Tn5 insertion and chemical mutagenesis. Specific mutation in the aggA locus by Tn5 insertion results in loss of agglutinability that is complemented in trans by a wild-type copy of the P. putida aggA locus. We examined the biochemical bases of agglutination in P. putida by comparing cell surface features in Agg+, Agg- mutants, and a genetically restored aggA mutant. No changes in gross cell surface features involving hydrophobic or hydrophilic binding or net negative charge were observed. Three macromolecular features, pili, flagella, and lipopolysaccharide size, did not differ between Agg+ and Agg- mutants. Protein profiles of cell envelope, periplasmic, and outer membrane preparations revealed pleiotropic effects of mutation in agglutination phenotype including alterations of an outer membrane protein of 47,000 molecular weight and periplasmic proteins of 56,000 and 60,000 molecular weight. The protein alterations seen in the aggA::Tn5 Agg- mutant 5123 reverted to wild-type patterns upon introduction of a wild-type copy of the aggA locus. These data suggest agglutinability may be conditioned by more than one proteinaceous component associated with the bacterial envelope layers.

[Congenital macrodactyly]. / Congenitalis makrodaktilia.

Macrodactyly is a rare congenital anomaly affecting the limbs. A case--treated by author at Al Thawra Hospital Sanaa, Yemen Arab Republic--is presented with a brief review of the literature and the difficulties of the treatment.

Fusarium Wilt Suppression and Agglutinability of Pseudomonas putida.

Mutants of Pseudomonas putida (Agg) that lack the ability to agglutinate with components present in washes of bean and cucumber roots showed limited potential to protect cucumber plants against Fusarium oxysporum f. sp. cucumerinum. However, a higher level of protection was observed against Fusarium wilt in cucumber plants coinoculated with the parental bacterium (Agg), which was agglutinable. The Agg mutants did not colonize the roots of cucumber plants as extensively as the Agg parental isolate did. In competition experiments involving bean roots inoculated with a mixture of Agg and Agg bacteria, the Agg strains colonized roots to a greater extent than the Agg cells did. These data suggest that the Agg phenotype provides additional interactions that aid in the beneficial character of P. putida.

Molecular Studies on the Role of a Root Surface Agglutinin in Adherence and Colonization by Pseudomonas putida.

Pseudomonas putida aggressively colonizes root surfaces and is agglutinated by a root surface glycoprotein. Mutants of P. putida derived chemically or by Tn5 insertion demonstrated enhanced or decreased agglutinability. Two nonagglutinable Tn5 mutants (Agg) and two mutants with enhanced agglutinability (Agg) possessed Tn5 in unique restriction sites. Agg mutants colonized root surfaces of seedlings grown from inoculated seeds, but at levels lower than those observed with the Agg parent. In short-term binding studies, Agg cells adhered at levels that were 20- to 30-fold less than those for Agg parental cells. These data suggest that the agglutination interaction plays a role in the attachment of P. putida to root surfaces.