DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.     

Intron 1 sequences are required for pancreatic expression of the human proglucagon gene

The mammalian proglucagon gene is expressed in pancreatic islet A-cells, intestinal L-cells, and select neurons of the brain, where posttranslational processing results in the liberation of a unique profile of peptides. Despite the importance of proglucagon-derived peptides in human biology, little is known about the regulation of the human gene, as the rat gene has been the preferred model for understanding the regulation of proglucagon gene expression. Previously, we have shown that although the immediate promoter region of the rat proglucagon gene is sufficient for expression in pancreatic islet cells, the homologous human proglucagon promoter sequences are not sufficient. We have now used a comparative genomic approach to identify noncoding sequences near the human proglucagon gene that are conserved among mammals, and thus potentially are regulatory sequences. Our alignments identified three evolutionarily conserved noncoding regions (ECR), one is the immediate promoter region (ECR1), the second is about 5 kb 5' to the mRNA start site (ECR2), and the third is near the 3' end of the first intron (ECR3). Our in vitro transient transfection assays with reporter gene constructs that include the human ECR3 support expression in rodent islet cell lines. Complementary studies with transgenic mice possessing a reporter gene regulated by a human proglucagon gene promoter-intron 1 (including ECR3) sequences express the reporter gene in the pancreas, as well as the intestine and selected neurons. These studies suggest that conserved sequences within intron 1 of the human proglucagon gene are important for expression in the pancreas.     

Adenovirus sequences required for replication in vivo.

We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occupies the first 18 to 21 bp and includes sequences conserved between all adenovirus serotypes. The adjacent auxillary region extends past nucleotide 36 but not past nucleotide 67 and contains the binding site for nuclear factor I.     

Location of additional early gene sequences in the adenoviral chromosome.

Genetic and biochemical analysis has revealed additional early gene sequences in the group C human adenovirus genome. Physical mapping by an improved marker rescue method showed that a group of "early function" mutations previously found by us to map within the left quarter of the genome do not map within the known early sequence (0-11.2) in that region, but instead lie within the coordinates 18.5-22.0. A search for early expression in this region of the genome using saturation hybridization techniques showed the presence of two discrete sets of viral cytoplasmic RNA sequences, present early in infection at low copy number and complementary to sequences on the I strand of the adenovirus genome lying between 11.2-14.5 and 19.8-23.5 untis.     

Effect of c-myc gene expression on early inducible reactions required for erythroid differentiation in vitro.

By employing cell fusion between two genetically marked mouse erythroleukemia (MEL) cells in which an artificially introduced c-myc gene had been placed under the control of human metallothionein promoter, we investigated the mechanism of the suppressive action of c-myc gene expression in erythroid differentiation. The results indicated that the expression of the c-myc gene blocked the induction of dimethyl sulfoxide-inducible activity, one of the two early activities required for triggering the differentiation.     

Cloned DNA sequences that determine mRNA stability of bacteriophage phi X174 in vivo are functional.

The stability of two species of phi X174 polycistronic mRNA in vivo can be altered by mutating sequences existing immediately upstream of a termination site. The wild type phage contains an mRNA stabilizing sequence ((+) sequence), while the same sequence mutated by insertion ((-) sequence) reduces the stability of the mRNAs. These two sequences were cloned at the 3' ends of gene D or gene B of phi X174 in a pBR322 derivative plasmid. The cloned sequences were functional. The (+) sequence stabilized gene B or gene D mRNA; half-lives of these mRNAs were 7 to 8 min. When the (+) sequence is eliminated ((o) sequence) or replaced with the (-) sequence, the half-lives of the mRNA were reduced to about 1 to 2 min. The stabilization of mRNAs caused an increased production of these proteins.     

cis-acting sequences that control the level of viral DNA synthesis in the polyomavirus late region.

A deletion in the polyomavirus late region results in a drastic reduction of viral replication, as shown after transfection of viral DNA into 3T6 cells. This mutation is cis acting, since cotransfection with wild-type DNA did not restore the normal phenotype. Viral DNA synthesis returned to normal levels only after reintroduction of the authentic sequences in either orientation. The data presented here suggest that these sequences are involved in the binding of a factor(s) that controls the level of viral replication.