[Generation and analysis of novel mutations of the trithorax-like gene in Drosophila melanogaster].

The Trithorax-like (Trl) gene of Drosophila melanogaster encodes the multifunctional GAGA factor. The expression of Trl is known to depend on numerous factors, such as the organ, the tissue, the ontogenetic stage, and the ambient temperature. Apparently, this expression is controlled by a complex system of regulatory elements, which so far has been scarcely studied. Our preliminary results indicate that the second intron of the Trl gene bears functionally significant elements. To test this assumption, we generated 23 novel alleles of the gene via P-induced male recombination and analyzed them cytogenetically. Of these mutations, 13 (recessive lethals) are deletions, disrupting the coding gene region. Ten mutations (seven deletions and three duplications) remove parts of the second Trl intron only. Some of these mutant stocks exhibit lower viability at different temperatures. These results suggest that the second intron region harbors functionally significant elements. The deletion mapping results verified the localization of the Trl gene in the 70F1-2 region.

Insect globin gene polymorphisms: intronic minisatellites and a retroposon interrupting exon 1 of homologous globin genes in Chironomus (Diptera).

Exon 1 of globin gene ct-13RT in clone lambdagb2-1 from Chironomus thummi contains a 444nt SINE (CTRT1). Based on in situ hybridization to polytene salivary gland chromosomes, C. thummi (ct), C. piger (cp) and C. tentans (ctn) contain copies of CTRT1 at multiple chromosomal loci. Genomic PCR amplifications reveal interrupted (ct-13RT) and uninterrupted (ct-13) alleles of the globin gene in the German population of C. thummi maintained in our laboratory, and only uninterrupted alleles or their homologs in different populations of C. thummi, C. piger and C. tentans. PCR amplification did generate different length fragments from cp-13 gene homologs in natural and laboratory C. piger populations that were due to variation in the length of minisatellite expansions of the central introns of the genes rather than a CTRT1-like SINE. Within minisatellite arrays, aligned homologs were more similar than paralogs in a single population, indicating that a tandem cluster of these repeats predated separation of the C. piger populations. The ct-13 genes of several C. thummi populations lack the minisatellites, suggesting their origin in C. piger only after the thummi/piger split. CTRT1 transposition into a ct-13 allele is even more recent, occurring after separation of German and other European C. thummi populations. The nearly intact ct-13RT and comparison with its intact ct-13 allele support a very recent transposition of the CTRT1 SINE into one of at least two already diverged ct-13 globin gene alleles. PCR analysis of DNA from individual adults in C. thummi shows a 1:2:1 distribution of ct-13/ct-13:ct-13/ct-13RT:ct-13RT/ct-13RT genotypes, consistent with a neutral spread of the ct-13RT allele since transposition, and indicating that the hemoglobin encoded by ct-13 is not necessary for survival, at least in a laboratory population of C. thummi.

The Chironomus (Camptochironomus) tentans genome contains two non-LTR retrotransposons.

A cDNA library from salivary gland cells of Chironomus tentans was screened with a probe containing the NLRCth1 non-LTR (long terminal repeat) retrotransposon from Chironomus thummi. Several positive clones were obtained and one of them, p62, was characterized by in situ hybridization and sequencing. The sequencing analysis showed that this clone contained a 4607 bp nucleotide sequence of a new transposable element that hybridized in situ to more than 100 sites over all four C. tentans chromosomes. The detailed analysis of this sequence revealed the presence of the 3'-end of open reading frame 1 (ORF1), a complete ORF2, and a 1.3-kb 3'-end untranslated region (UTR). The new element has been designated NLRCt2 (non-LTR retrotransposon 2 from C. tentans). A comparison of the nucleotide sequences of NLRCth1 and NLRCt2 showed 30% similarity in the region of ORF1 and 70% similarity in the region of ORF2. Based on the results of Southern blot analysis, two transposable elements have been found in the C. tentans genome, one of which is identical to NLRCth1 from C. thummi. This may be explained by horizontal transmission. The second element, NLRCt2, has been found in two different forms in the C. tentans genome. These can be distinguished by the presence of the 1.3-kb 3'-end UTR in one of the forms. Since the cDNA clone investigated was isolated from a tissue-specific cDNA library, the data showed that NRLCt2 is expressed in somatic cells.

Centromeric heterochromatin and satellite DNA in the Chironomus plumosus species group.

Species of the Chironomus plumosus group display significant differences in their amount of centromeric heterochromatin. A tandem-repetitive satellite-like DNA has been isolated from C. plumosus. This DNA accounts for a major part of the centromeric heterochromatin. The DNA element has a Sau3AI restriction site ("Sau elements") and a monomer length of 165 or 166 bp. It is A-T rich (73%) and reveals a moderate DNA curvature, as shown by gel migration and computer analysis. The chromosomal localization and genomic organization of Sau elements were studied in 24 Chironomus species by in situ hybridization and (or) Southern analysis. The DNA is predominantly located in the centromeric regions of nine species, six from the plumosus group and three others. In some cases, inter- and intra-specific differences in the size of the centromeric heterochromatin seem to correlate with the strength of Sau element hybridization signals. Few species contain Sau repeats outside of the centromeres (C. borokensis, C. plumosus). Additionally, Sau elements are revealed to be present in ectopic threads between centromeres, and in B chromosomes found in C. borokensis and C. annularius.