In situ localization of the evolutionary conserved Cpy/Cty gene in the subfamily Chironominae (Chironomidae, Diptera): establishment of chromosomal homologies

The homologous sites on the salivary gland chromosomes of 13 species from three genera: Chironomus, Glyptotendipes, Kiefferulus have been mapped by means of fluorescent in situ hybridization using the evolutionary conserved gene Cpy/Cty (clone Cla1.1). In all species of genus Chironomus and genus Kiefferulus , the Cty/Cpy gene is located on arm F of chromosome EF. The relocation of the gene among the species of genus Chironomus can be done by simple or complex homozygous inversions which occurred during the divergent evolution of the chromosome of the species. In the genus Glyptotendipes , the Cty/Cpy gene was localized in arm E of chromosome EF. Since the banding patterns of salivary gland chromosomes between genus Chironomus and genus Glyptotendipes cannot be compared directly, in situ hybridization with clone of conservative gene was performed to be established some homologous chromosomes. The results obtained indicate that the chromosome arm F of Chironomus and chromosome arm E of Glyptotendipes may be homologous.     

Evolution of histone gene loci in chironomid midges.

In the present study we have localized the histone genes in the chromosomes of 16 different Chironomus species as well as in Prodiamesa olivacea, Glyptotendipes barbipes, and Acricotopus lucidus. In the genus of Chironomus we find four, five, or six different "major" chromosomal loci hybridizing with a histone gene cluster probe isolated from the genome of Chironomus thummi. These major histone gene loci probably contain clustered histone gene repeating units ("clustered" loci). They are located on one and the same chromosome arm in all but one of the species investigated. This shows that the histone gene clusters are rather conservative in their location over a long period of evolution. The comparison of the histone loci pattern from the chromosomes of the different chironomid species shows that there is good agreement with previously established chromosome maps and phylogenetic studies based on the chromosomal banding pattern. Stringent in situ hybridization with various histone gene containing clones suggest that the "clustered" histone gene loci are organized in a locus-specific way. In addition to the linked "clustered" histone gene loci, we found an isolated histone gene group ("orphon") present on chromosome IV in most Chironomus species. This gene group might be organized differently from the histone gene repeating unit described previously.     

Molecular identification of Chironomus spp. (Diptera) for biomonitoring of aquatic ecosystems

Abstract   Chironomidae larvae often represent a major component of the benthic fauna in inland water bodies and are used frequently as bioindicators of ecosystem health. The genus Chironomus is a recognised indicator of organic enrichment and has been used extensively in the northern hemisphere and New Zealand in ecotoxicological studies. However, similar use of Chironomus in Australia is limited due to the presence of cryptic species that restrict the collection of information on species-specific responses to environmental stress. To address the problems associated with species identification, we have used PCR-RFLP of the mitochondrial COI gene to develop DNA profiles for nine common Australian Chironomus species. Species-specific haplotypes were identified using reference taxa previously identified by cytological analysis, and verified with field specimens collected from seven wetlands around Melbourne. This research provides an effective tool for species identification of this ubiquitous and often abundant genus that will provide the basis of obtaining species-specific information to inform on the health of aquatic ecosystems.     

The nucleotide sequence and in situ localization of a gene for a dimeric haemoglobin from the midge Chironomus thummi piger

A cluster containing at least four globin genes was isolated by screening an lambda EMBL3 genomic DNA library of the midge Chironomus thummi piger (Ctp) with a heterologous haemoglobin (Hb) gene IV (HbIV) probe from Chironomus thummi thummi (Ctt). This globin gene cluster was localized by in situ hybridization to chromosome II. One globin gene together with its 5'- and 3'-flanking regions has been sequenced. It can be deduced from the sequence that it is a new member of the dimeric HbVIIB family. The Ctp HbVIIB-5 gene displays 91.8% nucleotide sequence homology to a HbVIIB cDNA sequence, reported previously. There is no evidence for intron/exon structure in the Ctp HbVIIB-5 gene.     

A Concertedly Evolving Region in Chironomus, Unique Within the Telomere

Chromosome terminal, complex repeats in the dipteran Chironomus pallidivittatus show rapid concerted evolution during which there is remarkably efficient homogenization of the repeat units within and between chromosome ends. It has been shown previously that gene conversion is likely to be an important component during these changes. The sequence evolution could be a result of different processes—exchanges between repeats in the tandem array as well as information transfer between units in different chromosomes—and is therefore difficult to analyze in detail. In this study the concerted evolution of a region present only once per chromosome, at the junction between the telomeric complex repeats and the subtelomeric DNA was therefore investigated in the two sibling species C. pallidivittatus and C. tentans. Material from individual microdissected chromosome ends was used, as well as clones from bulk genomic DNA. On the telomeric side of the border pronounced species-specific sequence differences were observed, the patterns being similar for clones of different origin within each species. Mutations had been transmitted efficiently between chromosomes also when adjoining, more distally localized DNA showed great differences in sequence, suggesting that gene conversion had taken place. The evolving telomeric region bordered proximally to subtelomeric DNA with high evolutionary constancy. More proximally localized, subtelomeric DNA evolved more rapidly and showed heterogeneity between species and chromosomes. Received: 24 September 1997 / Accepted: 24 November 1997     

The presence of 5 S RNA genes in two consecutive chromosome bands in Chironomus tentans

It has earlier been shown by in situ hybridization that the 5 S RNA genes are located in region 2A of chromosome II in Chironomus tentans [1]. In the present study the resolution in this chromosome region could be considerably improved by using the inversion 1 A/9C of chromosome II, which carries region 2A, not in the usual position close to one end of chromosome II, but in the middle of the chromosome. It was then revealed that electrophoretically isolated 5 S RNA hybridized in situ with two adjacent bands in the region 2A. It was also observed that in heterozygotes harbouring one normal chromosome II and one with the inversion 1 A/9C, there is a close physical contact between the bands containing the 5 S RNA genes and the nucleolar organizer region.     

The location of repeated DNA sequences in the chromosomes of Chironomus tentans

Polytene chromosomes of Chironomus tentans were hybridized in situ with in vivo labelled nuclear and chromosomal RNA. Nuclear RNA formed hybrids preferentially in five distinct regions considered to contain clustered, repeated DNA sequences. These are the two nucleolar organizer regions, Balbiani ring 1 and 2, and the 5 S RNA genes in region 2A of chromosome II, which together comprised almost 70% of the total number of grains over the complement. The remaining grains were diffusely distributed over the chromosomes. There was a significant difference in the distribution of grains when RNA from different chromosomes was used for hybridization. Chromosome I RNA hybridized preferentially with chromosome I, and chromosome II+III RNA preferentially with chromosome II+III. Some regions within the chromosomes hybridized significantly more chromosomal RNA than other regions. A considerable cross-hybridization of RNA from one particular type of chromosome with the other chromosomes was also found. It is concluded that repeated DNA sequences which hybridize with heterogeneous chromosomal RNA in C. tentans are widely dispersed in the genome. Some of these sequences have a delimited localization, others are dispersed, and some sequences which are transcribed in one particular chromosome are present also in the other chromosomes.     

Biochemische und cytogenetische Untersuchungen zur Natur des Hämoglobin-Polymorphismus bei Chironomus tentans und Chironomus pallidivittatus

The haemoglobin of chironomids is dissolved in the body fluid of the larvae and can be separated electrophoretically in Chironomus tentans into 10, and in C. pallidivittatus into 8, different bands. The molecular weight determined under the electrophoretic conditions was 15,000±1,000 for each haemoglobin band. This means that each haemoglobin band represented a single protein chain. In each species 7 haemoglobins could be characterised as species specific according to their different electrophoretic mobilities, developmental characteristics and the fact that one haemoglobin could be correlated genetically with a specific chromosome inversion. The inheritance of all these species specific haemoglobins was found to be co-dominant. With cytogenetic methods it was possible to define the loci of the species specific haemoglobin genes as being restricted to certain parts of chromosome 3. This finding suggests gene duplication as the most likely mecanism of the evolution of haemoglobins in Chironomus.     

Nuclear protein matrix of giant nuclei of polythene chromosome organization of midge <Emphasis Type="Italic">Chironomus plumosus</Emphasis> determinates

Giant nuclei from salivary glands of the midge Chironomus plumosus were treated in situ with 2M NaCl detergent and nucleases to reveal residual nuclear matrix proteins (NMP). It was shown that, after the prestabilization of nonhistone proteins with 2 mM CuCl₂, the polythene chromosome body preserved its morphologic integrity and banding pattern, even after the extraction of all histones and DNA. The stabilized NPM of polythene chromosomes can be observed in both light and electron microscopy; no interchromatin fibrillary-granular structures are revealed in the nucleus except for peripheral lamina. Using the immunocytochemical method, in polythene chromosomes, we managed to detect major nonhistone proteins (topoisomerase IIα and SMC 1) and some RNA-components. Besides, in giant nuclei of larvae of early stages there is observed BrDU incorporation visualizing sites of DNA synthesis, which also are connected with NPM of polythene chromosomes. Thus, it can be concluded that structure of NPM of giant nuclei of Chironomus plumosus has all properties of NPMs of usual interphase nuclei; furthermore, this NPM determines specific structure of the polythene chromosome.     

Localization of the mouse Mcf-2 (Dbl) protooncogene within a conserved linkage group on the mouse X chromosome

A mouse cDNA probe homologous to the human MCF2 transforming sequence has been identified and partially cloned, and is used here to localize the gene on the mouse X chromosome. The human gene has been physically mapped to within 60 kb of the gene for coagulation factor IX, within a large conserved linkage group between the mouse and human genomes which extends from HPRT to G6PD on the X chromosomes of both mammalian species. In situ hybridization of the mouse Mcf-2 probe onto mouse metaphase chromosomes indicates that this gene lies in the same region of the X chromosome as Cf-9, the mouse gene for coagulation factor IX. Moreover, segregation of species-specific genomic DNA polymorphisms for Mcf-2 and Cf-9 in a total of 203 individuals derived from two large interspecific mouse backcross populations (which are also segregating for 17 other X-linked molecular markers) demonstrates that the mouse genes are separated by only 0.5 +/- 0.5 cM. Despite this short distance we were able to order Mcf-2 and Cf-9 relative to one another and other genes in this region. The mouse gene order Hprt-Cf-9-Mcf-2-G6pd predicts a similar ordering of genes on the human X chromosome, a gene order which has only recently been demonstrated by physical mapping. Thus, the map location and linkage relationships of the Mcf-2 gene are similar in man and mouse, and this unique protooncogenic locus is part of a conserved linkage group on the mammalian X chromosome.     

The extracellular domain of the human interferon gamma receptor interacts with a species-specific signal transducer.

At least two species-specific gene products are required for signal transduction by interferon gamma (IFN-gamma). The first is the IFN-gamma receptor, which binds ligand with high affinity in a species-specific manner. The second is an undetermined species-specific signal transducer(s). To determine whether the human IFN-gamma receptor (hIFN-gamma R) interacts directly with this signal transducer(s) and, if so, with what functional domain(s), we constructed expression vectors for the hIFN-gamma R and three hybrid human-murine IFN-gamma receptors. The hybrid receptors contained the extracellular, human IFN-gamma (hIFN-gamma) binding domain of the hIFN-gamma R, either the human or murine transmembrane domain, and either the human or murine intracellular domain. The vectors encoding these receptors were stably transfected into two mouse cell lines, one of which (SCC-16-5) contains a single copy of human chromosome 21. The resulting cell lines were treated with hIFN-gamma, and murine major histocompatibility complex class I antigen expression was analyzed by immunofluorescence flow cytometry. All transfected cell lines lacking human chromosome 21 remained insensitive to hIFN-gamma. However, all four of the IFN-gamma receptors were able to signal when expressed in the cell line containing human chromosome 21. We conclude that the extracellular domain of the IFN-gamma receptor is involved not only in the species specificity of IFN-gamma binding but also in signalling through interaction with an as yet unidentified species-specific factor(s) encoded by a gene(s) on human chromosome 21.     

Genetics and evolution of the acute phase proteins in mice

Summary In mammals, a number of liver-derived plasma proteins, termed acute phase reactants, are induced during an inflammation response. We have studied genetic variation in the structure and expression of several of these proteins in a variety of inbred and wild-derived mice. In a genetic cross, electrophoretic polymorphisms for the two ₁-acid glycoproteins, AGP-1 and AGP-2, co-segregated in 58 backcross progeny, indicating that either a single gene or two tightly-linked genes on chromosome 4 encode the AGPs. In the same backcross, segregation of variation in haptoglobin structure showed that the gene encoding this acute phase reactant is on chromosome 8. Structural variation in serum amyloid A correlated with restriction fragment length polymorphisms in the Saa gene determined by Taylor and Rowe (1984). Analysis of a number of highly diverged species of mice indicated that AGP expression has undergone considerable modification during evolution of the Mus genus; this is associated with alterations in Agp gene organization, which may include species-specific amplification and/or deletion events.     

Comparison of Chironomus usenicus and Chironomus curabilis with species of the group plumosus (Diptera) inferred from the mitochondrial DNA Gene COI and by the polytene chromosomes banding pattern

The sequence of a 595-bp fragment of the mitochondrial COI gene was determined for the species Chironomus usenicus and Chironomus curabilis of the genus Chironomus. Phylogenetic reconstructions based on the analysis of the COI gene sequence coincide on the whole with cytogenetic data, permitting Ch. usenicus and Ch. curabilis to be regarded as members of the group plumosus. Chironomus usenicus and Ch. plumosus have identical COI gene sequences. Two hypotheses explaining this identity are considered: inheritance of mtDNA from one of the parental species during hybridogenesis and horizontal transfer of mitochondrial genes.     

Structurally divergent histone H1 variants in chromosomes containing highly condensed interphase chromatin

Condensed and late-replicating interphase chromatin in the Dipertan insect Chironomus contains a divergent type of histone H1 with an inserted KAP-KAP repeat that is conserved in single H1 variants of Caenorhabditis elegans and Volvox carteri. H1 peptides comprising the insertion interact specifically with DNA. The Chironomid Glyptotendipes exhibits a corresponding correlation between the presence of condensed chromosome sections and the appearance of a divergent H1 subtype. The centromere regions and other sections of Glyptotendipes barbipes chromosomes are inaccessible to immunodecoration by anti-H2B and anti- H1 antibodies one of which is known to recognize nine different epitopes in all domains of the H1 molecule. Microelectrophoresis of the histones from manually isolated unfixed centromeres revealed the presence of H1 and core histones. H1 genes of G. barpipes were sequenced and found to belong to two groups. H1 II and H1 III are rather similar but differ remarkably from H1 I. About 30% of the deduced amino acid residues were found to be unique to H1 I. Most conspicuous is the insertion, SPAKSPGR, in H1 I that is lacking in H1 II and H1 III and at its position gives rise to the sequence repeat SPAKSPAKSPGR. The homologous H1 I gene in Glyptotendipes salinus encodes the very similar repeat TPAKSPAKSPGR. Both sequences are structurally related to the KAPKAP repeat in H1 I-1 specific for condensed chromosome sites in Chironomus and to the SPKKSPKK repeat in sea urchin sperm H1, lie at almost the same distance from the central globular domain, and could interact with linker DNA in packaging condensed chromatin.     

B-Chromosomen beiChironomus

In the population “Herrenmühle” ofChironomus plumosus 11% of the individuals contain one supernumerary chromosome. This B-chromosome is present both in germ-line and somatic cells. — InChironomus melanotus 6% of the larvae of the population “Falkau” carry supernumerary chromosomes. These B-chromosomes cannot be found in all nuclei of testis and soma, their number varies between cells within the individual. In both species the B-chromosomes represent centromeric fragments of chromosome IV as can be shown by their structure and pairing behaviour. — The polytene B-chromosome ofCh. plumosus exhibits a banding pattern in the salivary gland nuclei. Furthermore it is able to form an additional nucleolus in the nuclei of the malpighian tubules. InCh. melanotus band structures can be seen only in the B-chromosome of malpighian tubules. The larvae ofCh. melanotus, carrying B-chromosomes, show heterochromatic bodies in the salivary gland nuclei, varying in number and size in the nuclei of the same gland. These bodies are interpreted to be polytenic B-chromosomes divided into subunits.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Differences in the number of nucleolus organizers in Chironomus tepperi shown by in situ hybridization

Two stocks of Chironomus tepperi could be isolated. One stock, N(IV)⁺, contains nucleolus organizers in chromosome I and IV, whereas the other one, N(IV)^–, shows only one nucleolus in chromosome I. It is demonstrated by in situ hybridization with radioactive rRNA that the absence of the nucleolus in chromosome IV of stock N(IV)^– is not related to an inactivation of the nucleolar DNA, as might have been suggested, but is due to the lack of ribosomal cistrons.