Biomedical subjects

H G Keyl

Publications and source records attributed to H G Keyl.

14 recordsLinked to original sources

PubMed2001

A complex containing at least one zinc dependent HeLa nuclear protein binds to the intronic (gaa)(n) block of the frataxin gene.

We analyzed HeLa nuclear proteins binding to the (gaa)(n) harbouring intron 1 of nine frataxin alleles and characterized the structures of the repeats. Fragments with blocks longer than (gaa)(9) form spontaneously different intramolecular H-y topoisomeres in linear state. The observed triplexes depend on the length of the repeat. Interruption of the perfectly repeated (gaa)(n) block entails two structural regions. At least two HeLa nuclear proteins bind to the (gaa)(n) fragments resulting in a distinct major retarded complex as revealed by EMSA. One of these proteins is zinc dependent. Importantly, the fragment harbouring (gan)(121) binds additional proteins. Protein binding appears to be locus specific, and the binding affinity was found to be not random. The affinities of the different target fragments varied by a factor of four. Binding affinities of the fragments were not obviously correlated to differences in the composition of the repeats. DNase I footprinting revealed only weakly protected binding regions, but multiple HS sites in the repeat regions of the fragments. These findings and the fact, that DNA conformers observed in EMSA and electron microscopical experiments bind proteins, lead to the assumption that the proteins recognize, both, B-DNA and triple helical structures, but with different affinity. Possible functions of the proteins are discussed in the context of transformation of triple helical structures into B-DNA and the pathogenesis of FRDA.

Alleles↗

PubMed1999

Protein binding to simple repetitive sequences depends on DNA secondary structure(s).

Simple repetitive DNA sequences are ubiquitous constituents of eukaryotic chromosomes. The properties of simple repeats generate increased interest as expansions of certain trinucleotide blocks cause human diseases. We studied protein binding and structural features of (gaa x ttc)n tracts e.g. in the polymorphic frataxin intron 1 and (gt)n(ga)m stretches from different HLA-DRB1 alleles in their original genomic environments. Electrophoretic mobility shift assays revealed that HeLa nuclear proteins bind to DNA fragments containing these simple repeat blocks. The major retarded protein/DNA complexes comprise, in both cases, zinc-dependent proteins present in nuclear extracts from different cell types. Competition experiments using various simple repeats differing in length and flanking regions demonstrate specific interactions. DNase I footprinting shows protein-binding sites located either within the repeats alone or within the repeats as well as their flanking regions, often with preference for one strand. Comparing different (gt)n(ga)m alleles, a regular pattern of footprints was not detectable in the (gt)n part indicating that the zinc-dependent protein recognizes structural rather than sequence-specific features. OsO4 and DEPC modifications followed by electrophoretic and electron microscopical analyses demonstrate that the homopurine blocks often form different types of intramolecular triple helices. A similar situation was evident using (gaa x ttc)n blocks of different lengths within frataxin intron 1 as targets. These data have functional implications for non-coding (gaa x ttc)n and (gt)n(ga)m tracts with regard to gene expression in vivo.

Animals↗

PubMed1998

A genome-derived (gaa.ttc)24 trinucleotide block binds nuclear protein(s) specifically and forms triple helices.

The properties of simple trinucleotide repeats generate increased interest as expansions of certain trinucleotide blocks cause human diseases. Here, we studied protein binding and structural features of a perfect (gaa.ttc)24 tract in its original genomic environment. Electrophoretic mobility shift assays revealed that HeLa nuclear proteins bind to the DNA fragment containing the (gaa.ttc)24 block. Competition experiments using simple (gt.ac)n repeats differing in length and flanking regions showed no cross-reactivity with the major retarded band. For the specific (gaa. ttc)n/protein complex, a binding constant of 9.3x10-9 mol/l was determined. DNase I footprinting revealed protein binding sites located exclusively within the repeat with a preference for the (gaa)24 strand. OsO4 and DEPC modifications followed by electrophoretic and electron microscopical analyses showed that the (gaa.ttc)24 block forms different types of intramolecular triple helices: Under superhelical stress, different H-DNA isomers are evident, whereas exclusively H-Y forms were detected in the relaxed state. Together, these data have functional implications for genomic (gaa.ttc)n tracts.

Base Sequence↗

PubMed1989

Comparison of insect hemoglobins (Erythrocruorins) from Chironomus thummi thummi and Chironomus thummi piger (Diptera). The primary structure of the monomeric hemoglobin CTP III.

The monomeric hemoglobin fractions of Chironomus thummi thummi (CTT) and Chironomus thummi piger (CTP) differ in the ratio of their components. The determination of the primary structure of the component CTP III was achieved by automatic Edman degradation of the native chain, the tryptic peptides and the C-terminal fragment, obtained by cleavage at the single tryptophan residue. It revealed two chains in the ratio 1:1 which share the ambiguity threonine/isoleucine in position 57 with CTT III. Whereas one chain is identical to the CTT III hemoglobin, the other differs in having isoleucine in position 105 and alanine in position 134. The CTP monomeric hemoglobin fraction comprises 8% of a component (CTP IV A) with a more negative charge than CTT IV but with an identical sequence up to position 44. This study reveals a very high polymorphism within Chironomus species and points out the need for more data at the gene level in order to provide better understanding of this striking phenomenon.

Amino Acid Sequence↗

PubMed1982

Cloning and analysis of ribosomal DNA of Chironomus thummi piger and Chironomus thummi thummi. The nontranscribed spacer of Ch. th. thummi contains a highly repetitive DNA sequence.

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, repetitive 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.

Animals↗

PubMed1981

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.

Animals↗

PubMed1975

Lampbrush chromosomes in spermatocytes of Chironomus.

In the primary spermatocytes of Chironomus pallidivittatus lampbrush chromosomes in pachytene and diplotene stages can be isolated by using a spreading technique.--In the earliest analyzable stages of the pachytene the lampbrush loops, although different in size, are identically structured, consisting of spiralized fibers covered with a thin matrix. These loops contain an average DNA length of 33 mum. They are connected by axial fibers of 6 nm in diameter at intervals of 0.15--0.07 mum without any interposing chromomeres. The axis is double-stranded.--On the basis of the DNA distribution patterns it is supposed that the loops contain those chromatid regions which are included in the bands of the polytene chromosomes of the same species. The lack of a typical RNP transcript in the loops and the finding that 98% of the total DNA is found in the loops lead to the conception that the lampbrush organization in these spermatocytes is primarily involved in the orderly course of crossing-over and in the preparation for the metaphase contraction of the chromatids.

Animals↗

PubMed1972