Intervening DNA sequences

The second NIST human DNA SRM is a PCR-based DNA Profiling Standard. The PCR was first described by Saiki et al. (1985,1989). Since then it has developed into a highly versatile and widely used detection, identification, manipulation and analysis tool in molecular biology, including DNA profiling. In brief, two short synthetic oligonucleotides, or primers, are used to define an intervening DNA sequence... 

Fig. 1.29. Mechanism of promoter activation of (/ -dependent genes in procaryotes. The formation of an open, initiation-competent transcription complex for (/ -dependent genes requires the assistance of transcription activators, which bind to their cognate UAS element. Upon loop formation of the intervening DNA sequences, the transcription activator interacts with the (/ -con-taing RNA polymerase bound to the promoter. The activation is accompanied by ATP hydrolysis and leads to the formation of an open complex.

Before the hnRNA produced by RNA polymerase II (see p. 242) can leave the nucleus in order to serve as a template for protein synthesis in the cytoplasm, it has to undergo several modifications first. Even during transcription, the two ends of the transcript have additional nucleotides added (A). The sections that correspond to the intervening gene sequences in the DNA (introns) are then cut out (splicing see B). Other transcripts—e.g., the 45 S precursor of rRNA formed by polymerase I (see p. 242)—are broken down into smaller fragments by nucleases before export into the cytoplasm. 

Fig. 1.19. Tetramerization of the Lac repressor and loop formation of the DNA. The Lac repressor from E. coli binds as a dimer to the two-fold symmetric operator seqnence, whereby each of the monomers contacts a half-site of a recognition sequence. The Lac operon of E. coli possesses three operator sequences Of, 02 and 03, aU three of which are required for complete repression. Of and 03 are separated by 93 bp, and only these two sequences are displayed in the figure above. Between Of and 03 is a binding site for the CAP protein and the contact surface for the RNA polymerase. The Lac repressor acts as a tetramer. It is therefore assumed that two dimers of the repressor associate to form the active tetramer, whereby one of the two dimers is bound to 03, the other dimer binds to Of. The intervening DNA forms a so-caUed repression loop. After Lewis et al., 1996.

The sequences of the recombination sites recognized by site-specific recombinases are partially asymmetric (nonpalindromic), and the two recombining sites align in the same orientation during the recombinase reaction. The outcome depends on the location and orientation of the recombination sites (Fig. 25-39). If the two sites are on the same DNA molecule, the reaction either inverts or deletes the intervening DNA, determined by whether the recombination sites have the opposite or the same... 

If recombination occurs within a piece of DNA at two homologous sites such as the attL and attR sites at the boundaries of the X prophage, the intervening DNA will be excised as a circular particle (Eq. 27-15). In this instance the two homologous regions must be repeated in the same direction, as is indicated by the arrow structures in Eq. 27-15. If the homologous sequences are oriented in opposite directions, i.e., they are inverted repeats, excision will not occur but the piece of DNA between the repeats will be inverted (Eq. 27-16). 

Fig. 1. In the germ-line (embryo) DNA, sequences coding for the variable (V) region lie distant from those encoding the constant (C) region. During the differentiation of B lymphocytes, these two sequences are brought together to form an active antibody gene by deletion of the intervening DNA (somatic recombination).

Since its introduction in 1977 this has become the most widely used sequencing procedure for DNA. In the intervening period the method has been refined and developed to the point where it has become the method of choice in laboratories all over the world. Though not as fast as the primed synthesis methods, it is, up to the time of writing the most widely tried and tested procedure and the majority of DNA sequences reported in the literature to date have been determined by the Maxam-Gilbert procedure. 

Figure 3. Examples of DNA sequences containing a stilbenedicarboxamide electron acceptor Sa. The rates of charge transfer between the primary G (green) and the distal GG doublet (red) strongly depend on the Intervening base sequence as explained in the text. For all sequences, hole transitions between G and GG are shown by arrows. Numbers next to the arrows are the values of rate constants for the hole transfer between G and GG. Corresponding average times for this process are given in parentheses.

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