Mutants sites

Xu J, Chen J, Toptygin D, Tcherkasskaya O, Callis PR, King J, Brand L, Knutson J (2009) Femtosecond fluorescence spectra of tryptophan in human gamma-crystallin mutants site-dependent ultrafast quenching. J Am Chem Soc 131(46) 16751-16757... 

Cold-adapted mutants Site-directed mutants Gene-deleted mutants Gene reassessment mutants Natiwally available mutants Mutants generated by adaptation to unnatural host... 

Fiq. 3. Map of mutant sites based on frequencies of am recombinants in N. crassa. Amino acid replacements of am (residue 141) and am7 (residue 142) cannot be aligned by recombination frequency (see text). 

Ultimately, this mutation results in half the daughter DNA duplexes being normal and half having a TA pair that had been CG. The first two rounds of replication at the mutant site -will be as follows ... 

A. and B. Constructs used for generation of the double-deletion psbDI7psbC7psbDir Synechocvstis 6803 strain which is the acceptor for constructs containing site-directed mutations in psbDI. C. Plasmid for reintroduction of psbPI/psbC into the double-deletion mutant. Site-directed mutations made in the construct shown in Fig. 2 can be introduced into this plasmid using suitable unique restriction sites in and near PsbDI. The length of the plasmids Is 7.9 kbp (A), 5.8 kbp (B), and 9.3 kbp (C). 

Initiator constitutive mutants have been isolated as revertants of a strain containing deletion 719, a deletion that excises araO and all known mutant sites in the araC gene. An original set of nineteen independent revertants was isolated on mineral L-arabinose agar plates subsequent to treatment of the culture with the mutagen diethyl sulfate. The revertants were detected on the plates after approximately 6 days of incubation at 37°C [11]. r mutations have also been induced with 2-aminopurine in this deletion mutant as well as in deletion mutant 766 [14],... 

Fig. 4. Structural genes araD, araA, and araB. Mutant sites indicated were previously ordered by three-factor reciprocal crosses. The order of a number of these sites, indicated by a heavy vertical line, has been confirmed by deletion mapping. Circled numbers indicate the site of mutations producing CRM. The stars indicate nonsense mutations. The dimensions in the figure do not represent the actual sizes of the gene.

Fig. 5. The regulatory gene, araC. The gene is divided into six sections by the deletions. The numbers above the small vertical line represent the site of C mutants ordered by three-factor reciprocal crosses and are placed at relative distances from one another based on recombination frequencies. (C/0/ to CI9 = 3.65 recombination units.) Other C mutant sites ordered by three-factor reciprocal crosses but without distance measurements are indicated only by a number (42,49,57). The order of several of the C mutant sites was confirmed by mapping with the deletion mutants indicated. mutants C 1-I0, C 60-67) isolated as resistant to o-fucose inhibition were mapped by recombination frequency. The position of mutant site C67, C 2, C4, and C 70 as being within the C gene was verified by deletion mapping. mutants (100 and over) isolated from a dK strain were ordered by deletion mapping. The star represents a nonsense mutation.

Constitutive mutants (C) for the L-arabinose pathway have been isolated from the wild type as mutants resistant to the D-fucose inhibition of growth on mineral L-arabinose D-fucose medium and from D-ribulokinaseless mutants dK ) as double mutants dK O) able to grow on mineral D-arabinose medium (see Sections II,B,1 II,B,2). The former C mutants were mapped by recombination frequencies [6]. With the availability of deletions ending at various positions within the araC gene, it became possible to map the C mutant sites using deletion mapping. All the C mutations isolated as dK C were mapped by this procedure [54] (Fig. 5). 

Of the 22 C mutants, isolated as resistant to D-fucose, 18 mapped between C" mutant sites. C8 and C 70 mapped close to C5. Both failed to give any wild-type recombinants with C5, and based on the number of Ara recombinants assayed, it was estimated that C 8 and 070 are, respectively, less than 0.02 and 0.1 recombination unit away from C5. The position of other mutant sites 067, 02, 04, 070) as being within the C gene was verified by deletion mapping [14]. 

It is interesting to note that the O mutant sites are not randomly distributed throughout the C gene, but are concentrated in the left half of the gene. There does not seem to be any pattern, as yet discernible,... 

Fig. 6. Controlling sites in the ara OJBAD operon. The deletions employed in proving the position of the controlling sites are indicated. represents the position of the / mutant sites. Mutant sites 901, 910, 911, 913, and 916 are absolute negatives, and it has not been determined whether they map at the beginning of araB, for instance at the AUG codon, or in the ara/region [27], Stars indicate the nonsense mutations in gene araB.

In nine out of ten cases, it was possible to cross, at low frequency, the P mutant sites into a strain containing deletion 1109, a deletion extending from araC to the leu operon. This placed nine Z mutant sites to the left of the deletion, and thus to the left of araC, in the region proposed to contain the initiator site for the L-arabinose operon. These... 

Besides these technical aggravations, there are basic obstacles in the use of reverse mutation systems. A true reverse mutation at the original mutant site requires a specific base-pair substitution, insertion, or deletion which need not be inducible by all mutagens. A very drastic... 

---