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.     

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.     

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.     

Nonreciprocal gonadal dysgenesis in hybrids of the chironomid midgeChironomus thummi. IV. Behavior of a female-specific protein,probably a vitellogenin

Using denaturing SDS-polyacrylamide gel electrophoresis, a protein with a subunit MW of about 148,000 daltons could be detected in the fat body of females of the reciprocal hybrids of Chironomus thummi thummi and Chironomus thummi piger, which is not present in males. This protein is presumably a vitellogenin and can be found in both hybrids during the late fourth-instar larval stage until eclosion of the adults, i.e., in early vitellogenesis. After eclosion, the reciprocal hybrids behave differently concerning the 148-kd protein. In females of the piger female x thummi male cross, which are fertile and produce yolky eggs, the 148-kd protein disappears from the fat body immediately after eclosion. In females of the reciprocal cross (thummi female x piger male) which are affected by gonadal dysgenesis and in which the oocytes only rarely contain yolk, the 148-kd protein is still present in the fat body of the adult up to 50 hr after eclosion until the fat body degrades. It is concluded that the inability of the sterile thummi female x piger male females to produce yolky eggs is caused by an impaired uptake of the presumed 148-kd vitellogenin into oocytes and not by a defective vitellogenesis. The impaired vitellogenin deposition into oocytes is taken as another aberrant trait of gonadal dysgenesis of the thummi female x piger male hybrids.     

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.     

Telomeres, interstitial telomeric repeat sequences, and chromosomal aberrations

Telomeres are specialized nucleoproteic complexes localized at the physical ends of linear eukaryotic chromosomes that maintain their stability and integrity. The DNA component of telomeres is characterized by being a G-rich double stranded DNA composed by short fragments tandemly repeated with different sequences depending on the species considered. At the chromosome level, telomeres or, more properly, telomeric repeats--the DNA component of telomeres--can be detected either by using the fluorescence in situ hybridization (FISH) technique with a DNA or a peptide nucleic acid (PNA) (pan)telomeric probe, i.e., which identifies simultaneously all of the telomeres in a metaphase cell, or by the primed in situ labeling (PRINS) reaction using an oligonucleotide primer complementary to the telomeric DNA repeated sequence. Using these techniques, incomplete chromosome elements, acentric fragments, amplification and translocation of telomeric repeat sequences, telomeric associations and telomeric fusions can be identified. In addition, chromosome orientation (CO)-FISH allows to discriminate between the different types of telomeric fusions, namely telomere-telomere and telomere-DNA double strand break fusions and to detect recombination events at the telomere, i.e., telomeric sister-chromatid exchanges (T-SCE). In this review, we summarize our current knowledge of chromosomal aberrations involving telomeres and interstitial telomeric repeat sequences and their induction by physical and chemical mutagens. Since all of the studies on the induction of these types of aberrations were conducted in mammalian cells, the review will be focused on the chromosomal aberrations involving the TTAGGG sequence, i.e., the telomeric repeat sequence that "caps" the chromosomes of all vertebrate species.     

Heat shock phenomena in Chironomus tentans

Incubation of 4th instar larvae of Chironomus tentans at elevated temperatures leads in salivary and Malpighian chromosomes to the appearance of 4–5 new puffs. Previously present puffs, particularly Balbiani rings in salivary chromosomes, become drastically reduced. The reactions of region IV-5C and Balbiani ring 1 and 2 in salivary glands are quantitatively analyzed. Statistically significant heat shock effects are observed already after 5 min and reach a maximum between 30 and 60 min. The effective temperature range is small (between 33 to 40 ° C) with an optimum at 37 ° C. Above 40 ° C, i.e., at overheat shock temperatures, heat shock reactions are suppressed. Larvae heat or overheat shocked for 1–7 h or 15–30 min, respectively, survive when returned to normal culturing temperatures. The recovery from heat shock of the puffing pattern occurs in two phases: a fast one (10–20 min) and a slow one (up to 5 h) sometimes separated by a period of backlash. Quenching of overheat shocked larvae does not result in a delayed heat shock reaction.     

Heat-shock phenomena in Chironomus tentans

Isolated salivary glands of fourth instar larvae of Chironomus tentans were incubated at different above-normal temperatures for various lengths of time. Size changes in Balbiani ring 2 (BR2) and in the heat-inducible chromosome region IV-5C were quantified. These chromosome regions behaved in vitro very much the same as in vivo: BR2 was repressed rapidly by heat shock (37° C), whereas under overheat-shock conditions (42° C) it stayed decondensed. Region IV-5C showed the opposite responses. After a return from heat shock to normal temperatures the puffing pattern recovered. This process depended strictly on the integrity of the gland and on a change of medium. In injured glands a recovery process occurring under heat-shock conditions was discovered.     

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.     

New foldback transposable element TFB1 found in histone genes of the midge Chironomus thummi

A new Foldback transposable element (TFB1) has been found in the histone H1-H3 intergenic region in the midge Chironomus thummi thummi. TFB1 has long terminal inverted repeats, composed of short, degenerate subrepeats and is flanked by nine or ten base-pair "target site" duplications. TFB1 is present in at least two adjacent histone gene units in Ch. th. thummi, indicating a homogenization of histone gene repeats. The copy number and chromosomal distribution of TFB1 are different in the closely related subspecies Ch. th. thummi and Ch. th. piger. showing that amplification, elimination and transposition of TFB1 have occurred recently during evolution.