Ziel
The aim of this proposal is to develop new methods and reagents for the four strategies explained in the Generalities and to evaluate their application for human genome analysis. The intentions are:
to screen for and isolate rare cutter nuclease and methylases from procaryotes;
to establish methods based on methylation-dependent restriction;
to search for new group l encoded endonucleases and establish their application;
to develop methods to chemically cleave DNA and optimize the resulting artificial nuclease for the specific cleavage of any complex DNA.
New tools were developed for cutting deoxyribonucleic acid (DNA) in the megabase range in order to improve the physical mapping of the human genome and to get access to specific regions (genes) of the human genome. New cleavage techniques are being developed which cut DNA at sequences greater than 12 base pairs in order to create tools o cut the human genome at any desired site. This involved development of an insertion technique using the intron encoded rare cutter endonuclease I-Scel, by purifying and analysis of another intron encoded rare cutter endonuclease I-CsmI and by development of artificial nucleases. New nucleases are being developed which recognize DNA sequences up to 12 base pairs. This involves screening for new restriction endonucleases recognizing 8 bp sequences and modification of restriction endonuclease activity by methylation of the substrate DNA.
Research with respect to the construction vectors and assessment of their properties involved the following:
cutting bacterial DNA yeast development of and mouse DNA with 3-SceI;
development of a new mapping strategy;
development of I-Csm I as a site specific endonuclease.
Using conventional and established nucleotide chemistry different nucleotide triphosphates coupled to orthophenanthroline (OP) via a spacer arm have been synthesized. The length of the spacer arm has been varied in order to achieve optimal incorporation and cutting. Enzymatic incorporation has been established with several DNA and ribonucleic acid (RNA) polymerases.
With respect to novel restriction endonucleases, a preliminary screen of 15 strains of bacteria has been completed and enzymes for further characterization have been identified. Strategies for producing rare cleavage of DNA have been done in 2 ways: combinations of restriction endonucleases and modification methylases to generate high specificity combinations of enzymes and secondly, protection of selected sites by DNA binding proteins (the 'Achilles heel' method).
Archilles heel cleavage was performed with Oct2A as binding protein, with MerR as binding protein and with Raf repressor as binding protein.
In order to analyze the structure of the human DNA and to clarify the mechanisms of specific human diseases many laboratories have recently focused on the development a of complete physical map of the human chromosomes with the ultimate goal of sequencing the entire human genome. Physical mapping, one of the key techniques of the human genome mapping project, has been greatly advanced by the invention of pulsed field gel electrophoresis. This technique was the prerequisite for fractionating and analyzing DNA molecules of sizes in the megabase range.
Megabase maps are crucial for human genome analysis since they fill the gap between the fine resolution physical map in the kb range, including sequencing, and the low resolution genetic (linkage) map in the cM range. However, megabase mapping has been limited so far by the low number of rare cutter nucleases which allow the fragmentation of human DNA at sites of 8 bp length or longer. Thus, the success of the human genome mapping project will depend greatly on the availability of new tools for megabase mapping.
The work will focus on the creation of appropriate technologies for the production of physical maps, in the megabase range, of all human chromosomes. So far four main strategies to fragment human DNA in the megabase range are known but only very few reagents are available to carry out any of these strategies:
restriction enzymes recognizing more than 6 bp; only Not I, SfiI, SgrAI, PacI, RsrII are commercially available;
combination of methylases and restriction enzymes which produce cleavage specificity up to 12 bp only are commercially available;
group I intron encoded endonucleases with up to 18bp recognition site; ISceI, not available yet;
artificial nuclease; none available.
Innovation:
New classes of tools for megabase mapping will be developed. New 8 bp restriction enzymes and combinations of methylases and nuclease resulting in up to 12 bp cutting sites will improve, facilitate and speed up many mapping strategies in the megabase range. Group I intron encoded 18 bp cutters will open up new possibilities for the precise fragmentation of whole chromosomes. Artificial nucleases may become multipotent tools to cut human DNA precisely at any desired site. This may allow one to define directly the distance between known genes and therefore improve physical mapping of the whole human genome tremendously.
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82377 Penzberg
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