DNA recombination

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

Two types of mutants have been used for amino add overproduction auxotrophic and regulatory mutants. In some cases, mutant strains have been further improved through DNA-recombination. 

Another way to enhance the production of an amino acid is to make use of DNA-recombinant technology, often in combination with foe mutations already described. In this way foe negative features of foe micro-organisms are avoided. To help explain this, we will consider a well known fermentation of L-phenylalanine using Escherichia coli. We have already seen foe metabolic pathway leading to foe production of L-phenylalanine in Figure 8.4. 

The most obvious alternative approach is to deregulate the aroF gene, which is subject to tyrosine regulation. This, of course, could be achieved by DNA-recombinant techniques or by mutation. 

Both the heat and cold shock response are universal and have been studied extensively. The major heat shock proteins (HSPs) are highly conserved. They are involved in the homeostatic adaptation of cells to harsh environmental conditions. Some act as molecular chaperones for protein folding, while others are involved in the processing of denatured polypeptides whose accumulation would be deleterious. The cold shock results in the transient induction of cold shock proteins (CSPs), which include a family of small acidic proteins carrying the cold shock domain. The CSPs appear to be involved in various cellular functions such as transcription, translation and DNA recombination. 

Abecassis, V., Pompon, D. and Truan, G. (2000) High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2. Nucleic Acids Research, 28, E88. 

Much of what we know about the regulation of information flow (gene expression) has been made possible by the ability to manipulate the structures of DNA, RNA, and proteins and see how this affects their function. The ability to manipulate DNA (recombinant-DNA methods) has generated a new language filled with strange-sounding acronyms that are easy to understand if you know what they mean but impossible to understand if you don t. Understand ... 

Another, promising avenue to understand silk protein conformation and assembly is the use of model peptides. Although not recent (Fraser and MacRae, 1973 Lotz et al., 1974), studies of silk-based peptide from chemical synthesis, DNA recombinant technology, and computer simulation (Anderson et al., 1994 Asakura et al., 2003 Fahnestock et al., 2000 Fossey et al., 1991 Heslot, 1998 Kaplan, 1998 Wilson et al., 2000) have shown that selected repeats of silk proteins can be transformable hydrogels, elastomers, or regular thermoplastics and that with a proper design they can function as diverse molecular machines (Altman et al., 2003 Heslot, 1998 Kaplan, 1998 Urry, 1998). 

Enzymes required in site-specific DNA recombination, moving transposable elements around in DNA molecules of bacteria and other organisms. 

Borggrefe T, Wabl M, Akhmedov AT, Jessberger R (1998) A B-cell-specific DNA recombination complex. J Biol Chem 273 17025-17035... 

Positive results from the Saccharomyces cerevisiae mitotic recombination assay indicate that a substance induces DNA recombination in this test system, i.e., mitotic gene conversion (within a gene) and/or mitotic crossing-over (between a gene and its centromere). 

Sauer B, Henderson N (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage PI. Proc Natl Acad Sci USA 85 5166-5170... 

The BRCAl and BRCA2 genes mediate a hereditary susceptibility for breast cancer. Both gene products bind to the human RadSl protein which is involved in DNA recombination and DNA repair. The BRCAl gene has been shown to be involved in the transcriptional control of genes that participate directly or indirectly in the repair of oxidative DNA damage (Gowen et al., 1998). 

Allergy and Infectious Diseases. Report on a workshop of DNA recombinant technology in interferon cloning. J Infect Dis, 1981.143 297-300. 

Re combinational DNA repair of a circular bacterial chromosome, while essential, sometimes generates deleterious byproducts. The resolution of a Holliday junction at a replication fork by a nuclease such as RuvC, followed by completion of replication, can give rise to one of two products the usual two monomeric chromosomes or a contiguous dimeric chromosome (Fig. 25-41). In the latter case, the covalently linked chromosomes cannot be segregated to daughter cells at cell division and the dividing cells become stuck. A specialized site-specific recombination system in E. coli, the XerCD system, converts the dimeric chromosomes to monomeric chromosomes so that cell division can proceed. The reaction is a site-specific deletion reaction (Fig. 25-39b). This is another example of the close coordination between DNA recombination processes and other aspects of DNA metabolism. 

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