Precise and imprecise somatic excision of the transposon Tc1 in the nematode C. elegans.

Eleven chromosomal products of somatic excision of Tc1 transposable elements have been cloned and sequenced. The cloning method did not involve genetic reversion; therefore the products analyzed should be representative. Six empty religated target sites were from excision of one Tc1 element inserted near actin genes on linkage group V; five were from a second Tc1 element inserted elsewhere on the same linkage group. All six products from the first element were identical in sequence to an empty target site from a second strain, indicating excision had been precise. Two of the products from the second element were also precise, whereas the other three contained four extra nucleotides at the point of excision, indicating an imprecise excision. The four nucleotides are the same in all cases and could represent two terminal nucleotides of the transposon plus a two-nucleotide target site duplication. The difference in the ratio of precise to imprecise excision at the two insertion sites suggests a possible chromosomal position effect on the pathway of Tc1 somatic excision.     

Identification of a Putative Structural Gene for Cathepsin D in Caenorhabditis Elegans

Mutants of Caenorhabditis elegans having about 10% of wild-type activity of the aspartyl protease cathepsin D have been isolated by screening. Mutant homozygotes have normal growth rates and no obvious morphological or developmental abnormalities. The mutant gene (cad-1) has been mapped to the right extremity of linkage group II. Heterozygous animals (cad-1/+) show intermediate enzyme levels and animals heterozygous for chromosomal deficiencies of the right extremity of linkage group II have 50% of wild-type activity. Cathepsin D purified from a mutant strain has a lower activity per unit mass of pure enzyme. These data suggest that cad-1 is a structural gene for cathepsin D.     

Nucleotide sequences of Caenorhabditis elegans core histone genes. Genes for different histone classes share common flanking sequence elements

We have determined the nucleotide sequence of core histone genes and flanking regions from two of approximately 11 different genomic histone clusters of the nematode Caenorhabditis elegans. Four histone genes from one cluster (H3, H4, H2B, H2A) and two histone genes from another (H4 and H2A) were analyzed. The predicted amino acid sequences of the two H4 and H2A proteins from the two clusters are identical, whereas the nucleotide sequences of the genes have diverged 9% (H2A) and 12% (H4). Flanking sequences, which are mostly not similar, were compared to identify putative regulatory elements. A conserved sequence of 34 base-pairs is present 19 to 42 nucleotides 3' of the termination codon of all the genes. Within the conserved sequence is a 16-base dyad sequence homologous to the one typically found at the 3' end of histone genes from higher eukaryotes. The C. elegans core histone genes are organized as divergently transcribed pairs of H3-H4 and H2A-H2B and contain 5' conserved sequence elements in the shared spacer regions. One of the sequence elements, 5' CTCCNCCTNCCCACCNCANA 3', is located immediately upstream from the canonical TATA homology of each gene. Another sequence element, 5' CTGCGGGGACACATNT 3', is present in the spacer of each heterotypic pair. These two 5' conserved sequences are not present in the promoter region of histone genes from other organisms, where 5' conserved sequences are usually different for each histone class. They are also not found in non-histone genes of C. elegans. These putative regulatory sequences of C. elegans core histone genes are similar to the regulatory elements of both higher and lower eukaryotes. The coding regions of the genes and the 3' regulatory sequences are similar to those of higher eukaryotes, whereas the presence of common 5' sequence elements upstream from genes of different histone classes is similar to histone promoter elements in yeast.     

Safe To Walk? Neighborhood Safety and Physical Activity Among Public Housing Residents

Background

Despite its health benefits, physical inactivity is pervasive, particularly among those living in lower-income urban communities. In such settings, neighborhood safety may impact willingness to be regularly physically active. We examined the association of perceived neighborhood safety with pedometer-determined physical activity and physical activity self-efficacy.

Methods and Findings

Participants were 1,180 predominantly racial/ethnic minority adults recruited from 12 urban low-income housing complexes in metropolitan Boston. Participants completed a 5-d pedometer data-collection protocol and self-reported their perceptions of neighborhood safety and self-efficacy (i.e., confidence in the ability to be physically active). Gender-stratified bivariate and multivariable random effects models were estimated to account for within-site clustering. Most participants reported feeling safe during the day, while just over one-third (36%) felt safe at night. We found no association between daytime safety reports and physical activity among both men and women. There was also no association between night-time safety reports and physical activity among men (p = 0.23) but women who reported feeling unsafe (versus safe) at night showed significantly fewer steps per day (4,302 versus 5,178, p = 0.01). Perceiving one''s neighborhood as unsafe during the day was associated with significantly lower odds of having high physical activity self-efficacy among both men (OR 0.40, p = 0.01) and women (OR 0.68, p = 0.02).

Conclusions

Residing in a neighborhood that is perceived to be unsafe at night is a barrier to regular physical activity among individuals, especially women, living in urban low-income housing. Feeling unsafe may also diminish confidence in the ability to be more physically active. Both of these factors may limit the effectiveness of physical activity promotion strategies delivered in similar settings.     

A monoclonal antibody recognizes an epitope common to an avian-specific nuclear antigen and to cytokeratins

X3, a monoclonal antibody of unusual specificity, is described. This antibody reacts with one or more cytokeratin polypeptides and also reacts with an avian (chicken, quail) nuclear antigen that appears to be present in all cell types (chicken) tested, although with variable staining pattern and intensity. This antigen is distinct from the cytokeratins but does have an epitope in common with this class of proteins. It disappears from the nucleus during the early stages of cell division and reappears during anaphase as a granular cytoplasmic structure. In late telophase the antigen is relocated in the nucleus. This antigen, which we have designated as avian-specific nuclear antigen (AVNA), is not associated with chromatin or ribonucleoproteins. From immunoblotting experiments on chicken fibroblast nuclei, AVNA is probably a complex composed of one or several polypeptides, one of which has a molecular weight of approximately 60 kD. The proteins were identified as nuclear matrix proteins rather than pore complex-lamina proteins by immunoblotting experiments on the purified nuclear matrix of chicken erythrocytes. The major polypeptide had a molecular weight of 60 kD and the minor polypeptide a molecular weight of 69 kD.     

Fine tuning of TCR signaling by CD5

Current data indicate that CD5 functions as an inhibitor of TCR signal transduction. Consistent with this role, thymocyte selection in TCR transgenic/CD5(-/-) mice is altered in a manner suggestive of enhanced TCR signaling. However, the impact of CD5 deletion on thymocyte selection varies depending on the transgenic TCR analyzed, ranging from a slight to a marked shift from positive toward negative selection. An explanation for the variable effect of CD5 on selection is suggested by the observation that CD5 surface expression is regulated by TCR signal intensity during development and CD5 surface levels on mature thymocytes and T cells parallel the avidity of the positively selecting TCR/MHC/ligand interaction. In this study, we generated mice that overexpress CD5 during thymocyte development (CD5-tg), and then examined the effect of CD5 overexpression or CD5 deletion (CD5(-/-)) on selection of thymocytes that express the same TCR transgenes. The results demonstrate that the effect on thymocyte selection of altering CD5 expression depends on the avidity of the selecting interaction and, consequently, the level of basal (endogenous) CD5 surface expression. Substitution of endogenous CD5 with a transgene encoding a truncated form of the protein failed to rescue the CD5(-/-) phenotype, demonstrating that the cytoplasmic domain of CD5 is required for its inhibitory function. Together, these results indicate that inducible regulation of CD5 surface expression during thymocyte selection functions to fine tune the TCR signaling response.     

Extrachromosomal circular copies of the transposon Tc1.

The 1.6 kb Tc1 transposable element of Caenorhabditis elegans undergoes excision and transposition in the germline. In somatic tissue it is excised at high frequency. Extrachromosomal linear and circular copies of Tc1 have been identified that are likely to be products of somatic and germline excision. In the present study, we have determined the sequences of the sites of circularization in circular extrachromosomal Tc1 molecules. DNA molecules containing these sites were cloned after PCR amplification with primers directed outward from within Tc1. Sequences were obtained with two complete Tc1 ends and one or more intervening copies of the TA dinucleotide, with one complete end and one deleted end, and with two deleted ends. The 24 clones had different structures, indicating the pool of molecules serving as PCR templates was heterogeneous. The predominant circular junction had one or more nucleotides deleted from at least one transposon end. Such a molecule without two complete ends might not be expected to serve as a transposition intermediate. Hence, some extrachromosomal circular Tc1 molecules may result from a deadend excision pathway.     

Receptor for retrograde transport in the apicomplexan parasite Toxoplasma gondii

Toxoplasma gondii and its apicomplexan relatives (such as Plasmodium falciparum, which causes malaria) are obligate intracellular parasites that rely on sequential protein release from specialized secretory organelles for invasion and multiplication within host cells. Because of the importance of these unusual membrane trafficking pathways for drug development and comparative cell biology, characterizing them is essential. In particular, it is unclear what role retrieval mechanisms play in parasite membrane trafficking or where they operate. Previously, we showed that T. gondii's beta-COP (TgBetaCOP; a subunit of coatomer protein complex I, COPI) and retrieval reporters localize exclusively to the zone between the parasite endoplasmic reticulum (ER) and Golgi apparatus. This suggested the existence of an HDEL receptor in T. gondii. We have now identified, cloned, and sequenced this receptor, TgERD2. TgERD2 localizes in a Golgi or ER pattern suggestive of the HDEL retrieval reporter (K. M. Hager, B. Striepen, L. G. Tilney, and D. S. Roos, J. Cell Sci. 112:2631-2638, 1999). A functional assay reveals that TgERD2 is able to complement the Saccharomyces cerevisiae ERD2 null mutant. Retrieval studies reveal that stable expression of a fluorescent exogenous retrieval ligand results in a dispersal of betaCOP signal throughout the cytoplasm and, surprisingly, results in betaCOP staining of the vacuolar space of the parasite. In contrast, stable expression of TgERD2GFP does not appear to disturb betaCOP staining. In addition to TgERD2, Toxoplasma contains two more divergent ERD2 relatives. Phylogenetic analysis reveals that these proteins belong to a previously unrecognized ERD2 subfamily common to plants and alveolate organisms and as such could represent mediators of parasite-specific retrieval functions. No evidence of class 2 ERD2 proteins was found in metazoan organisms or fungi.