Different repetition frequencies of a 120 base-pair DNA-element and its arrangement in Chironomus thummi thummi and Chironomus thummi piger

The repetition frequency of a highly repetitive DNA sequence has been measured in the genomes of Ch. thummi thummi and Ch. th. piger. This sequence is known to be involved in the evolutionary duplication of defined chromosomal segments leading to a significant increase in the genome size of Ch. th. thummi. Reassociation of this highly repetitive DNA sequence which has a repeat length of 120 base-pairs, with total Ch. th. thummi and Ch. th. piger DNA has shown that the repetition frequency in the Ch. th. thummi DNA is 5.5 fold higher than in Ch. th. piger. In both genomes a 120 base-pair sequence is present as tandemly repeated sequence as shown by Southern analysis.     

Different hybrid effects in reciprocal crosses betweenChironomus thummi thummi andCh. th. piger including spontaneous chromsome aberrations and sterility

Hybrids from reciprocal erosses between twoChironomus thummi thummi andCh. th. piger laboratory stocks show four abnormalities in comparison to the parental stocks. One cross direction (Ch. th. thummi x Ch. th. piger ) is characterized by chromosome aberrations, reduced hatchability and malformations, whereas in reciprocal hybrids both sexes are sterile. Sterility is the consequence of rudimentary or non developed gonads.InCh. th. thummi x Ch. th. piger crosses chromosome aberrations were analysed in salivary gland nuclei. These aberrations are all somatic in origin, and they are induced during the first 40 h of embryonic development, prior to the onset of polytenization. The chromosomes of both subspecies are equally affected. In all four chromosomes breaks occur preferentially at specific regions. Reduced hatchability and malformations are presumably caused by chromosome mutations because within egg-masses a correlation exists between the rate of salivary gland chromosome aberrations and the rates of hatchability and malformations.     

DNS-Replikationsmuster der Speicheldrüsen-Chromosomen von Chironomiden

The pattern of DNA-synthesis of the salivary gland chromosomes of Chironomus thummi thummi, Ch. th. piger, Ch. annularius, Ch. plumosus and Ch. melanotus was studied using H³-thymidine-autoradiography. Contrary to the previous conception the bands of the salivary gland chromosomes of Chironomus do not begin replication simultaneously. H³-thymidine incorporation in bands of high DNA content begins later than in bands with a lesser amount of DNA. This difference in time is very small in bands outside the kinetochore regions and not comparable to the asynchrony in replication of typical heterochromatin in the salivary gland chromosomes of Chironomus melanotus. Differences in the amount of DNA in homologous bands do not affect the onset of replication. — Bands of high DNA content are replicating during a longer time than those having less DNA. However, certain chromosome regions behave differently. In these regions bands of very low DNA content are synthesizing DNA during the whole replication cycle. Since no excessive increase of DNA could be observed in these regions it is supposed that in addition to the duplication of structural DNA an extra DNA is synthesized which disappears immediately from the chromosome. — At the end of the replication cycle in the salivary gland nuclei of the hybrid Chironomus th. thummi X Ch. th. piger a labeling pattern is found in the chromosomes of Ch. th. thummi which differs from that in the parental subspecies Ch. th. thummi.     

Analysis of expression of the gene from the BRa puff of Chironomus thummi encoding the low molecular weight secretory protein

The plasmid containing F6.2 gene from the BRa of Chironomus thummi within the pUR 292 vector was constructed. Chimeric protein containing beta-galactosidase-F6.2 polypeptide was produced in Escherichia coli BMH71-18. The protein obtained has immunological similarity with secretion protein of 67,000 D from Ch. thummi salivary gland. The gene for sp67 is active in main and in special lobe of the gland. Based on the data obtained and on the previous results of the authors, conclusion is made that BRa is a complex locus containing several types of genes.     

Localization of a male sex determining chromosome region in Chironomus thummi piger

Chironomus th. thummi x Ch. th. piger hybrid males were backcrossed with Ch. th. thummi females. The salivary gland chromosomes of both sexes of the backcross progeny were then studied in respect to their pairing behavior. Region D₃d-g of chromosome III showed sex specific pairing. It is concluded that within this D₃d-g region a male sex determiner of Ch. th. piger is located and that the male is the heterogametic sex.     

The HLE hybrid dysgenesis syndrome in the midgeChironomus thummi: Differences in the dysgenic potential between strains

After crossing experiments between twoChironomus subspecies the HLE hybrid dysgenesis syndrome occurs non-reciprocally in the F1 hybrids if the males of each of the fourCh. th. thummi strains (HL, So, G, Kr) are crossed to females ofCh. th. piger, strain E. In hybrid egg masses, differences in the strength of the abnormal trait reduced egg hatch were found between the differentpiger E ×thummi interstrain crosses. From the results it is concluded that eachthummi strain has a specific hybrid dysgenesis potential which determines the severity of the HLE syndrome in the hybrids. The HLE syndrome occurs also in the hybrids of crosses of differentCh. th. thummi strains. In these hybrids the syndrome is only obtained if the male parent descends from a strain which has a higher HLE hybrid dysgenesis potential than that strain from which the female parent is derived. These results demonstrate that the HLE syndrome is not restricted to the hybrids of thethummi × piger subspecies cross. In theCh. th. thummi interstrain crosses the hybrids of that cross direction are normal, which is reciprocal to the cross direction affected by the HLE syndrome. This is in contrast to thethummi × piger subspecies crosses, where the hybrids of one cross direction always show the HLE syndrome and the hybrids of the reciprocal cross direction the Rud syndrome. Thus, the present study also demonstrates that the HLE and Rud hybrid dysgenesis syndrome inChironomus are independent from one another.     

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.     

An AT-rich DNA component in the genomes of Chironomus thummi thummi and Chironomus thummi piger

A DNA fraction has been isolated from total Chironomus thummi thummi DNA which is discernible from the bulk Ch. th. thummi DNA by a lower thermal stability. In situ hybridizations with polytene salivary gland chromosomes of Ch. th. thummi and Ch. th. piger made localization of this DNA fraction possible. Hybridizations with bands which contain different amounts of DNA in the two subspecies indicate that the isolated DNA fraction mostly consists of those sequences which represent the genetical difference between thummi and piger.This paper is dedicated to Professor Dr. H. Bauer on the occasion of his 75th birthday     

Cloning and analysis of ribosomal DNA of Chironomus thummi piger and chironomus thummi thummi

The ribosomal DNAs from Ch. thummi piger and Ch. th. thummi were cloned and analysed by a variety of restriction endonucleases. Comparison of rDNA clones from the two subspecies revealed a considerable length difference: the length of the analysed rDNA cistrons is approximately 9.0 kb for Ch. th. piger and approximately 14.5 kb for Ch. th. thummi. The nearly 5 kb additional DNA in Ch. th. thummi is clearly located within the non-transcribed spacer region, and consists of AT-rich, reptitive DNA elements. These elements with a basic repeat length of approximately 120 bp, are arranged tandemly in stretches of up to about 50 identical copies, which are characterized by a cleavage site for ClaI restriction endonuclease. They are found only in the Ch. th. thummi rDNA clones and not in the Ch. th. piger clones. Southern hybridizations between cloned ribosomal DNA and centromeric highly repetitive DNA have shown that the ribosomal repetitive Cla-elements are closely related to a highly repetitive DNA sequence family, which is present in various chromosomal sites particularly the centromeres. Sequence analysis has revealed more than 90% homology between the ribosomal Cla-elements and the centromeric Cla-elements. — Since it is clear from cytological investigations that Ch. th. piger with the small rDNA repeating unit is the phylogenetically older subspecies, we postulate a transposition of Cla-elements into the nucleolar DNA during the evolution of Ch. th. thummi.     

Analysis of a repetitive DNA family of two closely related chironomids,Chironomus thummi thummi andCh. thummi piger (Diptera) by density-gradient centrifugation,melting analysis and restriction analysis

The DNAs of the two subspecies ofChironomus thummi, Ch. th. thummi andCh. th. piger, were investigated by CsCl density-gradient centrifugation, melting analysis and restriction analysis including Southern hybridization with AT-rich, highly repetitive DNA sequences. The melting analysis of density-fractionatedCh. th. thummi andCh. th. piger DNA has shown that thethummi DNA contains an early melting DNA fraction, which is enriched in the light fractions of the density gradient. The DNA fraction is also present inpiger DNA though in lower concentration. Restriction and Southern analysis of density fractionatedthummi andpiger DNA has revealed that there are two tandemly-repetitive DNA-sequence families that hybridize with this AT-rich, early melting DNA fraction. One sequence is characterized by anHae-III site and a basic repeat length of 130 ± 15 bp and the other by aCla-1 restriction site and a basic repeat length of 120 ± 4 bp. These sequences are present in much higher concentrations in the genome ofCh. th. thummi when compared toCh. th. piger, and are hence correlated to the higher DNA content of theCh. th. thummi genome.     

Clustered and interspersed repetitive DNA sequence family of Chironomus. The nucleotide sequence of the Cla-elements and of various flanking sequences

The nucleotide sequence of more than 30 cloned members of the clustered and interspersed repetitive Cla-sequence family present in the genome of various chironomids has been determined. In four cloned Cla-element clusters, the 5' and 3'-flanking sequences including the junctions between the Cla-element clusters and the flanking sequences were also sequenced. The repetitive Cla-elements, which are able to transpose under certain circumstances, have a monomer length ranging from 110 to 119 base-pairs, are very A + T-rich (greater than 80% A + T) and display numerous palindromic sequences. The Cla-elements are organized in small (4 elements) to medium-sized (greater than 30 elements) tandem repetitive clusters, which are dispersed over more than 200 sites of the chromosomes of Chironomus thummi thummi, including the non-transcribed spacer of the ribosomal DNA repeating unit. The tandem repetitive Cla-elements show anomalous behaviour during high-percentage polyacrylamide gel electrophoresis, indicating a bent or globular conformation. The flanking sequences are also repetitive, but the sequenced parts did not reveal any tandem repetitive arrangement. Near the junctions of the Cla-element clusters and the flanking sequences, short duplications are found, ranging from 5 to 12 bases, present in both sides of the Cla-element clusters. The Cla-elements might be involved in the hybrid dysgenesis phenomenon that is observed after crossings between the two subspecies Ch. th. thummi and Ch. th. piger.     

Molecular-cytogenetic characteristics of B-chromosomes in chironomid (Diptera, Chironomidae)

Morphological and molecular study of B-chromosomes of three Chironomus species (siblings Ch. borokensis and Ch. phumosus from plumosus group, and Ch. heterodentatus from obtusidens group) was carried out. Morphological similarity of B-chromosome banding pattern and telomer-centromeric region banding pattern of chromosome IV in Ch. borokensis was shown. Polytene B-chromosomes of Ch. borokensis and Ch. heterodentatus were microdissected, and their DNA was amplified using degenerate oligonucleotide primer polymerase chain reaction. Comparative analysis of the localization of homologous B-chromosome DNA sequences of A- and B-polytene chromosomes was made using in situ fluorescence hybridization. It has been shown that B-chromosomes in the studied species are composed mainly of repetitive DNA sequences homologous to sequences of centromeric and telomeric DNA of A-chromosomes, and also these of the mobile element NLRCthl. The B-chromosome DNA, homologous to sequences of DNA mobile element, was scattered on A-chromosomes (more than 100 sites). No ribosomal DNA repeats were identified in B-chromosome. Heterologous FISH of B-chromosome DNA to polytene A-chromosomes of Ch. thummi, a species lacking B-chromosomes, enabled us to reveal the presence of numerous sites homologous to DNA of B-chromosomes. These are mainly mobile element sites. An origin of B-chromosomes and peculiarities of their organization in chironomids are discussed.     

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.     

<Emphasis Type="Italic">Mytilus edulis</Emphasis> Core Histone Genes Are Organized in Two Clusters Devoid of Linker Histone Genes

Abstract Comparison of histone gene cluster arrangements in several species has revealed a broad spectrum of histone gene patterns. To elucidate the core histone gene organization in a mollusk, we have analyzed a Mytilus edulis genomic library and have isolated eight phage clones containing core histone genes. Analysis of insert DNA revealed that the core histone genes are arranged as regular gene repeats of all four core histones. The repeats do not contain linker histone genes. The clones are distributed into two groups of dissimilar repeated units with a common size of about 5.6 kb. The genes of each core histone class in the distinct repeats encode identical histone proteins and have comparable gene arrangements in the two repeat units. However, the intergenic sequences differ significantly. The core histone genes are organized as large clusters of about 100 repeats each. Previously, we have shown that the linker histone genes in M. edulis are also organized in a cluster of repeats of solitary H1 genes. Hence, this is the first case of a separate, clustered organization of both core and linker histone genes, repectively.     

The role of holocarboxylase synthetase in genome stability is mediated partly by epigenomic synergies between methylation and biotinylation events

Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to histones. Biotinylated histones are gene repression marks and are particularly enriched in long terminal repeats, telomeres and other repeat regions. The effects of HLCS in gene regulation are mediated by its physical interactions with chromatin proteins such as histone H3, DNMT1, MeCP2 and EHMT-1. It appears that histone biotinylation depends on prior methylation of cytosines. De-repression of long terminal repeats in biotin- or HLCS-deficient cell cultures and organisms is associated with genome instability.Key words: biotin, DNMT1, EHMT-1, genome stability, histone, holocarboxylase synthetase, MeCP2, methylation     

N-banding in polytene chromosomes of Chironomus and Drosophila

The N-banding patterns of the polytene chromosomes of Drosophila melanogaster, Chironomus melanotus, Ch. th. thummi and Ch. th. thummi x Ch. th. piger were studied. In Chironomus the polytene N-banding patterns correspond to the polytene puffing patterns. This is revealed by comparison of the puffing and N-banding patterns of identical chromosomes. Size and staining intensity of the N-bands reflect the size of the puffs as shown by puff induction. There is no evidence that the N-bands are also located in Chironomus heterochromatin or are restricted to the nucleolar organizer regions. In Drosophila the -heterochromatin is strongly N-positive, whereas the -heterochromatin, as well as the Chironomus heterochromatin is not N-banded. Contrary to Chironomus, the puffs in Drosophila polytene chromosomes do not give rise selectively to well stained N-bands. — The N-banding method is interpreted to stain specifically non-histone protein which is (1) accumulated in genetically active chromosome regions and (2) present in a specific type of heterochromatin (-heterochromatin of Drosophila).     

The nucleotide sequence of an unusual non-transcribed spacer and its ancestor in the rDNA in Chironomus thummi.

The nucleotide sequence of the non-transcribed spacer (NTS) in the ribosomal DNA (rDNA) of Chironomus thummi thummi and Chironomus thummi piger, including major parts of the external transcribed spacer, is described. The NTS of the two subspecies are very different in length, (thummi, 7 kb, piger, 2 kb); this is due to the insertion into the NTS of C.th. thummi of a large cluster of highly repetitive DNA sequences which are not present in the NTS of C. th. piger. The repetitive sequences, called Cla elements, are present in high copy number elsewhere in the genome of C. th. thummi and, in lower copy number, in the genome of C. th. piger in which they are mainly in the centromeric regions. Sequencing of the NTS of thummi and piger yielded information on the junctions between the Cla element cluster and the original NTS sequence, as well as on the sequence of the integration site before the transposition has occurred. The integration site is characterized by a dA cluster at the one end and a dT cluster at the other.