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A morphological temperature-sensitive mutant of Caenorhabditis elegans displays 2 overlapping temperature-sensitive periods, both occurring during the post-embryonic development. Data prove that these 2 phenotypes are controlled by the same locus and are not inherited as maternal factors. [ABSTRACT FROM AUTHOR]

The human alpha-globin complex lies at the tip of the short arm of chromosome 16. It comprises three functional globin genes (5'-zeta2-alpha2-alpha1-3'), the expression of which is strictly dependent on a positive regulatory element located 40-kb upstream, HS-40. This DNase I-hypersensitive site is the only known regulatory element displaying strong erythroid-specific enhancer activity within the human alpha-globin complex. How this enhancer activity is shared among different erythroid genes present in the same cluster without affecting the ubiquitous genes present within and around the complex is poorly understood. To address this issue, we used hybrid murine erythroleukemia cells containing a single copy of human chromosome 16 and targeted the insertion of different sequences downstream of HS-40 by recombinase-mediated cassette exchange. We thus demonstrate that (i). HS-40-mediated erythroid-specific activation of the alpha-globin genes is impaired solely by the insertion of a promoter sequence and not a coding sequence, unless it is methylated, and that (ii). the degree of transcriptional repression observed seems to be related directly to the transcriptional rate of the inserted promoter. Taken together, these results emphasize the importance of promoter sequences as the main targets for the activation mechanism of the human alpha-globin genes by HS-40.

The human alpha-globin gene complex includes three functional globin genes (5'-zeta2-alpha2-alpha1-3') regulated by a common positive regulatory element named HS-40 displaying strong erythroid-specific enhancer activity. How this enhancer activity can be shared between different promoters present at different positions in the same complex is poorly understood. To address this question, we used homologous recombination to target the insertion of marker genes driven by cytomegalovirus or long terminal repeat promoters in both possible orientations either upstream or downstream from the HS-40 region into the single human alpha-globin gene locus present in hybrid mouse erythroleukemia cells. We also used CRE recombinase-mediated cassette exchange to target the insertion of a tagged alpha-globin gene at the same position downstream from HS-40. All these insertions led to a similar decrease in the HS-40-dependent transcription of downstream human alpha-globin genes in differentiated cells. Interestingly, this decrease is associated with the strong activation of the proximal newly inserted alpha-globin gene, whereas in marked contrast, the transcription of the non-erythroid marker genes remains insensitive to HS-40. Taken together, these results indicate that the enhancer activity of HS-40 can be trapped by non-erythroid promoters in both upstream and downstream directions without necessarily leading to their own activation.

We report the case of a normal individual displaying an extremely unbalanced G gamma/A gamma-globin ratio (G gamma-globin chains undetectable by urea/triton/ acrylamide gel electrophoresis and just reaching the threshold of detection by high performance liquid chromatography) associated with a very low level of G gamma-globin mRNA (at the most 5% of total gamma-mRNA after reverse transcriptase polymerase chain reaction determination). By DNA Southern blotting and sequencing, the very low level of G gamma-globin chains in this individual was found in association with subhaplotype [+ -----] (Hinc II 5' to epsilon, Xmn I 5' to G gamma, Hind III in G gamma and A gamma, Hinc II in and 3' to psi beta), with G gamma- and A gamma-globin gene sequences of the B type chromosome, and with a number of AT repeats in the locus control region hypersensitive site-2 site, similar to that reported to be associated with the Bantu beta S haplotype. These structural characteristics, described for the first time combined in the same individual, suggest that the G gamma/A gamma ratio in adults, is controlled by sequences distributed all along the beta-globin gene cluster.

We replaced the 3' flanking region of the human alpha 1-globin gene that binds in vitro the specific transcription factors GATA-1 and AP1/NF-E2, by a neo marker gene using homologous recombination in a MEL (mouse erythroleukemia line) hybrid cell line harbouring a single human chromosome 16. Using an improved method of the neo-positive and HSV-tk negative selection, one correctly targeted clone was obtained out of 164 clones analyzed. In contrast to non-targeted clones, the expression of teh neo gene in the targeted clone acquired the erythroid differentiation-dependent inducibility normally characteristic of the alpha-globin genes. No difference was observed in the expression of the human zeta, alpha 2, alpha 1, or theta-globin genes before and after induction of differentiation between the targeted clone and parental cells. These results indicate that, at least in the experimental system used, the 3' flanking region of the human alpha 1-globin gene can be replaced by an exogenous non-erythroid gene without affecting the regulation of the globin genes contained in the alpha-globin cluster.

The receptor for human macrophage colony stimulating factor (CSF-1R) was introduced into hematopoietic cell lines of myeloid and T-lymphoid origin, both of which normally do not express the CSF-1R. Infection of an interleukin-3 (IL-3)-dependent mouse myeloid cell line (FDC-P1) with a high titer retroviral vector expressing the human c-fms c-DNA, enabled CSF-1-dependent proliferation in short-term liquid culture assays as well as in clonal culture systems. CSF-1-dependent cell lines could be established after sorting for CSF-1R positive cells. In contrast to FDC-P1 cells, expression of the CSF-1R in CTLL cells, an IL-2-dependent mouse cytotoxic T-cell line, and in T-cell growth factor III/P40-dependent helper T-cells, ST2/K9.4a2, did not lead to CSF-1-dependent proliferation. These observations lead to the conclusion that ectopically expressed CSF-1R may function on certain myeloid cells where it is normally not expressed, suggesting the presence of signal transduction pathways which can be utilized by that foreign receptor. In contrast, it appears that T-lymphoid cells lack such a signalling mechanism, indicating that quite different modes of transducing mitogenic signals from the cell membrane to the nucleus must have developed during myeloid and T-lymphoid differentiation.

Collagen is present in Porifera, the lowest multicellular animals, but there is no information available on the primary structure of the collagen chains in this phylum. Developing fresh-water sponges have been used to extract total RNA in order to study in vitro translation products and to construct a cDNA library. Four translated proteins were collagenase-sensitive (200 kDa, 160 kDa, 81 kDa and 48 kDa). The cDNA library was screened with a human collagen probe and a clone, EmC4, covering 1.2 kb was isolated. Nucleotide sequencing of EmC4 revealed a conceptual open reading frame coding for 366 amino acids terminated by a stop codon TGA with 103 nucleotides downstream. The presumed translation product encoded contained several domains: a non-collagenous C-terminal domain of 156 amino acids with 9 cysteines, an uninterrupted collagenous domain of 171 amino acids, a non-collagenous domain of 16 amino acids with 3 cysteines and a probably incomplete N-terminal collagenous domain of 23 amino acids. Comparison with other sequences suggested that this collagen chain might belong to a non-fibrillar collagen family which evolved into several sub-families giving rise to nematode cuticular collagens, and type IV collagens.

Proteins of purified cuticles from adults of the small free-living nematode Caenorhabditis elegans are solubilized by reduction in the presence of a strong denaturing agent and then carboxymethylated. As in the large parasitic nematode Ascaris lumbricoïdes, these soluble proteins appeared to be collagens by their amino acid compositions. C. elegans cuticle collagen is separated into seven major components with different apparent molecular weights by molecular sieve chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The two main components, which together account for more than 64% of the total cuticle collagen, were extracted from gel after electrophoresis and analyzed. They differ in their amino acid compositions and would seem to represent genetically distinct collagen chains. The results presented lead to the hypothesis of the presence in this collagen of at least two different chains.

We have previously described the first homozygous cases of Hb Knossos in an Algerian family. The Hb A2 was completely absent, ascertaining the presence of a delta zero-thalassemia determinant in cis of the beta Knossos S gene. Here, we investigate the affected delta-globin gene. The complete DNA sequence of the gene and its 5' and 3' flanking regions was determined. Only two nucleotide changes were recorded: a C----T substitution at -199 and an AT insertion at -448 upstream from the cap site. To examine the involvement of these changes in gene function, the delta-gene was subcloned in an expression vector and introduced into COS cells. Analysis of RNA derived from these cells, using an S1 protection assay and dot-blot hybridization, revealed qualitatively and quantitatively normal transcription. The loss of delta-globin gene activity in vivo may be due to the alteration of a tissue-specific control.

The distribution of sites of type I collagen gene expression was studied in frozen sections of skin of 4 and 9 month-old calf fetuses by in situ hybridization using a human pro-alpha 1 type I collagen cDNA. The labelling varied with the different layers of the dermis and with the developmental stage considered. In the 4 month old fetus skin, the label appeared concentrated in the upper layer of the dermis at the lewel of the hair follicles. In the 9 month-old fetus skin, the difference of labelling between upper papillary dermis and lower dermis was less marked. Comparatively the distribution of the extracellular type I collagen was determined by indirect immunofluorescence. This collagen appeared present throughout the whole dermis with slight variations at 4 months, where there was less extracellular collagen near the hair bulbs. These results are in agreement with the idea that the collagen synthesis follows cutaneous differentiation. In addition, they support the hypothesis that collagen is deposited once morphogenetic events have occurred and plays thus a stabilizing role in formation of cutaneous appendages.