Huey-Lang Yang

Synthesis of the arabinose operon regulator protein in a cell-free system.

SummaryA DNA-directed cell-free system which synthesizes L-ribulokinase coded by the L-arabinose operon, ara OIBAD, has been developed. L-arabinose is required for the expression of this operon and, in addition, cyclic AMP and guanosine-tetraphosphate are needed for optimal synthesis. The ara C gene product is also required and can be supplied either by de novo synthesis in the cell-free system or added back from extracts of whole cells. Evidence is presented that the C gene product is highly susceptible to protease attack.

Involvement of arginine in in vitro repression of transcription of arginine genes C, B and H in Escherichia coli K 12.

Abstract The combination of L-arginine and partially purified arginine repressor (the argR gene product) represses transcription of argCBH in vitro .

A possible termination factor for transcription in Escherichia coli

DNA from both λdlac and λplac viruses direct mRNA-mediated synthesis of β-galactosidase (β-gal) in a coupled cell-free system. When λdlac DNA is used the system is almost completely dependent upon the presence of adenosine 3′,5′-cyclic monophosphate (cAMP) which is required for normal initiation of transcription at the lac promoter. By contrast when λplac DNA is used about half the maximum amount of β-gal is synthesized in the absence of cAMP. It seems highly likely that this cAMP independent synthesis of β-gal is the result of read-through from a phage initiation site on the λplac DNA. Since there is believed to be at least one transcription termination signal before the lac operon it must be asked why this signal is not being recognized as such in vitro. We hypothesize that the normal bacterial termination factor is missing or inactive in the bacterial extract used to make the cell-free system. This view is supported by the finding that protein extract can be isolated from whole E. coli cells under mild conditions which prevents the cAMP-independent β-gal synthesis without inhibiting cAMP-dependent β-gal synthesis. The behavior of the active factor (called the ϱy factor) in this extract is different from either the Roberts ϱ factor (called ϱR here) or the Schafer-Zillig κ factor. Furthermore it has been found that the ϱR factor cannot replace the ϱy factor.

In vitro synthesis and repression of argininosuccinase in Escherichia coli K12; partial purification of the arginine repressor.

SummaryΦ80dargECBH DNA has been used to direct cell-free synthesis of argininosuccinase, the argH gene product in Escherichia coli K12. In vitro enzyme synthesis is sensitive to repression by partially purified preparations from an argR+ strain but not by corresponding preparations from an argR- strain. Using DNA-cellulose chromatography, approximately seventyfold purification of repressor has been obtained. The partially purified preparation represses argininosuccinase synthesis but has no effect on β-galactosidase synthesis.

Nonradioactive DNA Hybridization: Application to Clinical Microbiology

While nucleic acid hybridization has been used to great benefit by molecular biologists, the application to clinical use has been limited. This is due to the requirement to label probe DNA with phosphorus-32 (32P). Since this isotope has a short half-life (14.1 days) and emits a high energy beta particle, 32P-labeled probes are short-lived and require special handling conditions and licensing. The recent development of nonradioactive DNA labeling procedures (1) presents the opportunity to apply the benefits of nucleic acid hybridization to routine clinical use. Diagnostic procedures based on this technology are being developed for clinical microbiology.

Regulation of Bacterial Genes

It is now apparent that the activities of many bacterial genes are regulated by several interlocking control apparatuses that respond to different small molecule effectors. An overview suggests the existence of a hierarchy of controls (Zubay, 1973). For example, the gene cluster known as the lac operon is induced by the presence of lactose. Lactose is also the substrate of the enzyme β-galactosidase, which is encoded by the lac operon. The induction of the lac operon by lactose is brought about by a highly specific control process that does not directly affect the activities of other genes. At a more general level of control, the expression of the lac operon is dependent upon the presence of 3’:5’-cyclic AMP (cAMP). This dependence is characteristic of many genes that encode catabolic enzymes. At an even more general level of control, we find a mechanism that appears to modulate transcription according to the gross rate of translation. The small molecule effector for this control process is probably guanosine tetraphosphate (guano-sine bis-diphosphate, ppGpp). The effect this compound has depends upon the gene — for the lac operon ppGpp is stimulatory.