In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities.

A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities. A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn-18, Ala-15, Val-14, Ser-11, and Arg-10. These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5'-A5T4T3G2C1G1'C2'A3A4'T5'-3'. Mutants containing the sequence Leu-18Tyr-15Xaa-14Tyr-11Arg-10, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5'-ATCYCGY'GAT-3' (Xaa = asparagine when Y = G; Xaa = methionine when Y = A). Moreover, in a selection against the sequence 5'-ATTACGTAAT-3', mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions. The mutant proteins showed high specificity in a functional transcription interference assay. A model for the interaction of these mutants with the target DNA sequences is discussed.

Cortical activity flips among quasi-stationary states.

Parallel recordings of spike trains of several single cortical neurons in behaving monkeys were analyzed as a hidden Markov process. The parallel spike trains were considered as a multivariate Poisson process whose vector firing rates change with time. As a consequence of this approach, the complete recording can be segmented into a sequence of a few statistically discriminated hidden states, whose dynamics are modeled as a first-order Markov chain. The biological validity and benefits of this approach were examined in several independent ways: (i) the statistical consistency of the segmentation and its correspondence to the behavior of the animals; (ii) direct measurement of the collective flips of activity, obtained by the model; and (iii) the relation between the segmentation and the pair-wise short-term cross-correlations between the recorded spike trains. Comparison with surrogate data was also carried out for each of the above examinations to assure their significance. Our results indicated the existence of well-separated states of activity, within which the firing rates were approximately stationary. With our present data we could reliably discriminate six to eight such states. The transitions between states were fast and were associated with concomitant changes of firing rates of several neurons. Different behavioral modes and stimuli were consistently reflected by different states of neural activity. Moreover, the pair-wise correlations between neurons varied considerably between the different states, supporting the hypothesis that these distinct states were brought about by the cooperative action of many neurons.