Henry B. Lowman

Temperature-mediated regulation and downstream inducible selection for controlling gene expression from the bacteriophage λ pL promoter

We have examined in detail the effects of various induction temperatures on the expression of a heterologous fusion gene controlled by the bacteriophage lambda PL promoter in a heat-inducible Escherichia coli expression system which utilizes the CIts857 repressor. Experiments performed over a temperature range spanning 29-42 degrees C indicate that, under our conditions, temperatures as low as 29 degrees C may be required to fully repress the CI857-controlled transcription from PL, and that the highest protein yields are obtained after induction at 36 degrees C for 6 h. We cloned the cat reporter gene downstream from a heterologous gene controlled by PL and found that cat expression at a low induction temperature permits the monitoring of productive transcription through the heterologous gene and thus aids in selecting transformants that are capable of producing the heterologous protein in E. coli.

Locations of nucleosomes on the regulatory region of simian virus 40 chromatin.

We have asked where the nucleosomes are located with respect to the replication origin and regulatory region of simian virus 40 DNA, what would be the possible functional consequences of the identified locations, and to what extent these locations correlate with the current views on mechanisms involved in establishing nucleosome-free regions in chromatin. To identify the precise location of nucleosomes, we have shot-gun cloned and sequenced nucleosomal DNA obtained from micrococcal nuclease digestion of wt776 chromatin prepared late in infection. Our results indicate that nucleosomes do not occupy unique positions over the replication origin or the elements involved in transcriptional control. However, it appears that the nucleosome distribution is not random, since several nucleosomes are represented by two or more independently generated clones. Two nearly identical cloned fragments map over the replication origin; five include 1.5 copies of the 72 base-pair enhancer sequences; and eight map to a region that spans a DNA bending locus and the major transcription initiation site of the late genes. The complex nucleosome distribution pattern observed in our direct analysis suggests that disparate nucleosome-free regions may be involved in controlling replication, and selective expression of the viral early or late genes.

SV40 Chromatin Structure and Virus Assembly

The process of virion assembly plays an active role in modulating the structure of SV40 chromatin. Experiments with temperature-sensitive mutants of the major capsid protein (VP1) have shown that minichromosomes which contain an exposed regulatory region accumulate when the initiation of SV40 assembly is blocked. VP1, directly or through specific interactions with other proteins, alters the average spacing between nucleosomes and may be involved in generating a protected regulatory region in SV40 chromatin. The ENA sequences near or between positions 640 and 875 interact with capsid proteins in vivo and appear to contain signals used for the initiation of shell assembly. A ENA topoisomerase is encapsidated during virion assembly. This enzyme is probably responsible for the difference in SV40 ENA linking number, observed previously, between unencapsidated chromatin and virion derived chromatin.

High-level expression of the simian virus 40 genes LP1, VP1 and VP2 as fusion proteins in Escherichia coli.

The complete sequences of the SV40 agnogene (LP1) and the genes coding for the capsid proteins VP1 and VP2 have been cloned into Escherichia coli expression plasmids. High levels of expression were obtained when the SV40 genes were inserted into the coding sequence of the influenza virus NS1 gene, which has previously been expressed in E. coli. The NS1A-LP1 and NS1A-VP2 chimeric proteins consist of the 81 N-terminal residues of NS1 (designated as peptide NS1A) fused to the complete sequence of the corresponding SV40 protein. The NS1A-VP1 chimera consists of NS1A followed by a linker of nine arbitrary residues and the complete sequence of the SV40 major capsid protein. The observed levels of expression vary considerably among the three chimeric proteins, ranging from approx. 70 micrograms/ml in the case of NS1A-LP1 to approx. 5 micrograms/ml in the case of NS1A-VP2. Cyanogen bromide cleavage of the NS1A-LP1 fusion protein produces fragments with Mrs expected for isolated NS1A and LP1 peptides. A plasmid has also been constructed which expresses the NS1A peptide in high yield.