Erwin R. Schmidt

In situ localization of two haemoglobin gene clusters in the chromosomes of 13 species of Chironomus

Two haemoglobin (Hb) gene clusters cloned from the genomic DNA of Chironomus thummi were localized in the polytene chromosomes of 13 Chironomus species. The haemoglobin gene cluster containing the genes for the monomeric haemoglobin proteins III and IV (λCttG1) hybridized in all species to the end of chromosome arm E. The haemoglobin gene cluster containing the genes for the dimeric HbVIIB proteins (λpiHb1) could be localized to chromosome arm D. The chromosomal position of the haemoglobin genes was always found in morphologically specified groups of bands and is in agreement with cytological data, which have been used to establish the evolutionary relationships between the species of the genus Chironomus.

Satellite DNA of Drosophila nasuta nasuta and D. n. albomicana: localization in polytene and metaphase chromosomes.

The DNA from the two Drosophila nasuta races, D. n. nasuta and D. n. albomicana was investigated by CsCl density gradient centrifugation. D. n. nasuta has one major AT-rich satellite DNA sequence with a density of 1.664g/cm3, while D. n. albomicana has at least three satellites with densities of 1.674g/cm3, 1.665g/cm3 and 1.661 g/cm3. The isolated satellite sequences hybridize in situ to all heterochromatic regions of all metaphase chromosomes of both races. In polytene chromosomes the satellite sequences hybridize exclusively to the chromocenter. All chromosomal regions hybridizing with the satellites show also bright quinacrine fluorescence.

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.

The development of a 120 basepair repetitive DNA sequence in Chironomus thummi is correlated to the duplication of defined chromosomal segments

In the dipteran species C’hironomus thummi, two subspecies exist which differ in their genome size by -30%. If chromosomes of Ch. th. thummi, the subspecies with the higher DNA contentlgenome, are compared with chromosomes of C’h. th. piger, one can find that numerous chromosomal bands of Ch. th. thummi are thicker than homologous bands of Ch. th. piger. Cytophotochemical measurements of the DNA content of single chromosomal bands have shown, that the thicker bands contain always 2” times (n = 1,2,3,4) as much DNA as the homologous bands in Ch. th. piger chromosomes [ 11. This has been interpreted as an evolutionary DNA increase by DNA duplication series. The analysis of the DNA of the two subspecies has revealed another difference between Ch. th. thummi and Ch. th. piger: an early melting AT-rich DNA subfraction is present in the 01. th. thummi DNA which is not detectable in Ch. th. piger DNA. This DNA fraction (-10% of the total DNA) called ‘80°CthDNA’ according to its melting point contains -30% highly repetitive DNA sequences. In situ hybridizations with polytene chromosomes of Ch. th. piger X thummi F1-hybrids have shown that the 80°C-thDNA is located in these chromosomal bands which are known to have the increased DNA content. Furthermore, in Ch. th. thummi these sequences are also present in chromosomal sites, where cytologically no duplication can be recognized. In Ch. th. piger, however, these sequences are present in a very low concentration and hybridize only to the centromeres of all 4 chromosomes [2]. The highly repetitive portion of the 80”C-th-DNA

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.

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.

Two AT-rich satellite DNAs in the chironomid Glyptotendipes barbipes (Staeger)

Two AT-rich satellite DNAs are present in the genome of Glyptotendipes barbipes. The two satellites have densities of 1.680 g/cm3 (=21% GC) and of 1.673 g/cm3 (=13% GC) in neutral CsCl-density gradients. The main band DNA has a density of 1.691 g/cm3 (=32% GC). This value is in agreement with the 33% GC-content of G. barbipes DNA calculated from thermal denaturation (TM=83° C). — In brain DNA as well as in salivary gland DNA the two satellite sequences together comprise 12–15% of the total G. barbipes DNA. Comparisons of the density profiles of DNA extracted from polytene and non-polytene larval tissue gave no hints for underreplication of the satellite DNAs during polytenization. — The two satellite DNAs have been isolated from total DNA by Hoechst 33258-CsCl density centrifugation and then localized in the polytene salivary gland chromosomes by in situ hybridization. Both satellite sequences hybridize to all heterochromatic centromere bands of all four chromosomes of G. barbipes. Satellite I (1.673 g/cm3) hybridizes mainly with the middle of the heterochromatin, satellite II (1.680 g/cm3) hybridizes with two bands at the margin of the heterochromatin. In situ hybridization with polytene chromosomes of Chironomus thummi revealed the presence of G. barbipes satellite sequences also in the Ch. thummi genome at various locations, mainly the centromere regions.

In situ binding of AT-rich repetitive DNA to the centromeric heterochromatin in polytene chromosomes of chironomids

Native highly repetitive DNA sequences have been allowed to react in situ with DNA-depleted polytene chromosomes of chironomids in cytological preparations. The double-stranded DNA can bind specifically to the centromeric heterochromatin, where these sequences have been localized previously by in situ hybridization. Various control experiments support the conception that heterochromatin-specific DNA-binding proteins are involved in the in situ binding.