[Study on gene knock-out in Mycobacterium BCG].

OBJECTIVE: To establish the methodology of plasmid for gene knock-out in Mycobacterium BCG. METHODS: We designed two pairs of primers for amplification of MDP1 gene and inserted two fragments into pKO plasmid, and then the recombinant plasmid for MDP1 gene knock-out was obtained, and named pKO-MDP1. Gene exchange took place within the genome of BCG after pKO-MDP1 plasmid was transformed into Mycobacterium BCG. The strain of Mycobacterium BCG with MDP1 gene knock-out was selected, and the curve of growth rate was studied. RESULTS: The target strain was that of the positive strain by two step PCR and one step sucrose counter selection, without growth in culture media with hygomycin. The value of A(600) per 12 hours was detected for sixteen days. A "S" shaped growth curve was detected. However, there was no significant difference in the growth rates between the wild type Mycobacterium BCG strain and the gene knock-out Mycobacterium BCG strain. CONCLUSIONS: The plasmid of pKO was a useful tool for gene knock-out in Mycobacteria. MDP1 maybe one of the factors influencing the growth rate, but it was not the only one.

Nonlinear dynamics of regulation of bacterial trp operon: model analysis of integrated effects of repression, feedback inhibition, and attenuation.

The trp operon encodes the five genes for the enzymes required to convert chorismate to tryptophan, and its switching on and off is controlled by both feedback repression and attenuation in response to different levels of tryptophan in the cell. Repression of the operon occurs when tryptophan concentration is high, and attenuation fine-tunes the transcription level at a lower cellular concentration of tryptophan. An extended mathematical model is established in this study to describe the switching on and off of the trp operon by considering the integrated effects of repression and attenuation. The influences of cell growth rate on the biosynthesis of tryptophan, stability and dynamic behavior of the trp operon are investigated. Sustained oscillations of tryptophan levels are predicted from the regulated turning on and off of the trp operon. It is interesting to note that during such oscillations the regulation of transcription displays a kind of "on" and "off" state in terms of gene expression, indicating the existence of a genetic circuit or switch in the regulation of the trp operon. Time lags between transcription and translation are also predicted and may explain the occurrence of such oscillation phenomenon.

Stem cell pluripotency and transcription factor Oct4.

Mammalian cell totipotency is a subject that has fascinated scientists for generations. A long lasting question whether some of the somatic cells retains totipotency was answered by the cloning of Dolly at the end of the 20th century. The dawn of the 21st has brought forward great expectations in harnessing the power of totipotentcy in medicine. Through stem cell biology, it is possible to generate any parts of the human body by stem cell engineering. Considerable resources will be devoted to harness the untapped potentials of stem cells in the foreseeable future which may transform medicine as we know today. At the molecular level, totipotency has been linked to a singular transcription factor and its expression appears to define whether a cell should be totipotent. Named Oct4, it can activate or repress the expression of various genes. Curiously, very little is known about Oct4 beyond its ability to regulate gene expression. The mechanism by which Oct4 specifies totipotency remains entirely unresolved. In this review, we summarize the structure and function of Oct4 and address issues related to Oct4 function in maintaining totipotency or pluripotency of embryonic stem cells.