Hans Zachau - RESEARCH ACTIVITIES#
tRNA structure and interactions, protein biosynthesis, 1957-1981#
My work on tRNA started in 1957 during a potdoctoral year in F. Lipmann's laboratory in New York and continued in Germany until the second half of the seventies, with last publications in 1981. In 1957 tRNA was a newly isolated RNA fraction and some of its basic features still had to be discovered. We were fortunate to find the site at which amino acids are bound to the RNA, i.e. the 3’-terminal adenosine, and to characterize the bond as a reactive ester bond (7).I started independent research work by choosing for myself and the first coworkers a ‘safe’ project, i.e. the study of model compounds for the reactive aminoacyl-tRNA esters and a ‘risky’ one, i.e. attempts at tRNA fractionation. Since 1960 our emphasis was on the isolation of specific tRNAs and subsequent structural work. This led, in 1966, to the elucidation of the nucleotide sequence of tRNASer from yeast (40), second to Holley's tRNA-Ala sequence of 1965. These were the very first nucleic acids to be sequenced which meant that, in the course of the work, the methods of sequencing had to be worked out. The tRNA-Ser sequence has been confirmed later at the DNA level, which cannot be said about all early tRNA sequences.
The known primary structures of several tRNAs served in the following years as the basis of biochemical studies on their conformations, on tRNA-synthetase interactions and on the mechanism of tRNA aminoacylation. Numerous tRNA fragments were prepared and combined by reassociation. These were early attempts to define those parts of the molecule which are important for its functions and/or for the maintenance of its threedimensional structure (54, reviews 57, 127). tRNA-synthetase and tRNA-ribosome interactions were studied also by physicochemical methods, frequently in collaboration with other labs (e.g. 112, 145).
Since 1969 the efforts of our group were divided between work on the relatively simple tRNA systems and the more complex systems of the eukaryotic genome and chromatin.
Repetitive DNA, 1970-1984#
The repetitive DNAs turned out to be interesting in their own right and useful for introducing to our laboratory the emerging cloning, mapping and DNA sequencing methods. Structural studies on several simple and complex satellite DNAs allowed conclusions as to the evolution of this class of DNA (e.g. 122, 152). Studies on middle and low repetitive DNAs yielded insight into genomic rearrangement processes (e.g. 187, 194).Chromatin, 1970-1984#
The chromatin work of our group started with a series of frustrating experiments on the then popular so-called chromosomal RNA which supposedly contained dihydrouridine residues as we knew them from our tRNA work; however we found the chromosomal RNA to be an artifact (69). The following studies were more rewarding; they dealt with the mechanisms of histone-DNA binding (e.g. 111), with nucleosome phasing on satellite DNA (review 172) and with the structure of chromatin domains (130). The chromatin structures at expressed and non-expressed immunoglobulin k genes of mouse were compared (e.g. 167, 196). A general review of chromatin research was written in 1982 (176).The studies on tRNA, tRNA-ribosome interactions, satellite DNA and chromatin were pursued independently by former coworkers.
Immunoglobulin genes, since 1977#
The work on the chromatin structure at the expressed and non-expressed immunoglobulin k genes led us to investigate the genes themselves. Germline and rearranged k genes of the mouse were cloned and sequenced. The finding of certain sequence elements, later called signal joints, contributed to the understanding of the mechanism of V-J rearrangements (158). Other early results concerned the problem of allelic exclusion (166) and the generation of antibody diversity by somatic mutations being restricted to the rearranged V genes and their near neighborhood (170). The discovery of the immunoglobulin gene promoters (192) led, in several laboratories including ours, to extensive studies on the expression of these genes.In 1981 we embarked on an attempt to elucidate the structure of the human k locus. The Ck , the 5 Jk and very few Vk gene segments were known at the time. We reached the goal in 1995 (reviews 260, 265,276). The V, J and C genes were cloned in l phages, cosmids and, in the last years, also in yeast artificial chromosomes. Contigs were constructed by chromosomal walking and a physical map of the locus and its surroundings was determined by pulsed field gel electrophoresis. The gene regions, recombination breakpoints and other regions of interest were sequenced. Our clones were recently sequenced by a Japanese group (277; see "Human k locus 2001").
The human k locus emerged as a 2 Mb structure which contained, in addition to the C and J genes, 76 V genes and pseudogenes, 40 of them in the so-called JC-proximal copy and 36 in a distal copy (review of sequences 258). Most JC-proximal V genes are rearranged by a deletion mechanism, the distal genes by an inversion mechanism (238). Somatic hypermutation as monitored in the transcripts, i.e. in the cDNAs, and in the k chain proteins affected the V genes in zero up to more than 10% of the positions (255). In addition to the Vk genes of the locus 25 so-called Vk orphons were found on different chromosomes (206 and review 260). Medical aspects included collaborative work on the gene(s) of the light chains of Haemophilus influenzae antibodies (review 245). Work on the structural variation of the k locus among individuals revealed a haplotype in which the 36 distal Vk genes are deleted (e.g. 241). Studies on the k locus of non-human primates yielded information on the evolution of the k genes; chimpanzees for instance possess only the proximal copy of the k locus, indicating that the duplication of the locus was a rather recent event (263).
In 1992 we resumed the study of the k genes of the mouse, the main experimental animal in immunology. The organization of the Vk genes within the locus is rather different from the one in the respective human locus. We found 140 Vk genes and pseudogenes in the locus and established its size to be close to 3.2 Mb, of which 3.1 Mb were cloned in four contigs. Apparently the mouse has twice as many Vk genes and a locus three times as large as that of man (270 - 273; 275, 276). By now three little gaps are still open in the locus. They will be closed at the latest, once the whole mouse genome has been sequenced some years from now.
A review on the k genes of human and mouse was published (No 278 in Publication List).
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