# GENETIC AND BIOCHEMICAL ANALYSIS OF AD5 REGION 2

> **NIH NIH R01** · ST. JUDE CHILDREN'S RESEARCH HOSPITAL · 1992 · $149,573

## Abstract

The objective of the work described in this proposal is to gain an
understanding of the control mechanisms operative in the regulation of
eukaryotic gene expression, using adenovirus type 5 (Ad5) early region 2A
as the model system.  The gene product of early region 2A is a 529 amino
acid single-stranded DNA binding protein (DBP) that is involved in viral
DNA replication, the host range of the virus, virus assembly, the
transcription of early region 4 (E4) and the stability of mRNAs from early
regions 1A and 1B.

The immediate goal of the proposed work is to precisely define within the
DBP domains that are involved in DNA binding and DNA replication.   Three
regions have been identified to date, and it is possible that other regions
of the protein are also involved.  Each region that is identified will be
subjected to saturation mutagenesis to define the contribution of
individual amino acids to the function of the DBP in DNA binding and DNA
replication.  The mutant DBPs will be assayed for their ability to
participate in the initiation and elongation reactions of adenovirus DNA
replication in vitro.

DBP mutants will be used to examine the role of the protein in controlling
transcription from the E4 promoter and the stability of mRNAs from E1A and
E1B.  Steady-state levels of RNAs will be measured by Northern blot
analysis, and rates of synthesis and decay of mRNAs measured by nuclear
run-on and continuous labeling techniques, respectively.

A possible role for the DBP in autoregulation will be explored by examining
the affinity of the protein for homologous and heterologous mRNAs, and its
effect on the in vitro translation of these RNAs.  RNAs will be synthesized
in vitro, binding to various regions of E2A and other mRNAs measured by
filter binding assays, and the effect of the DBP on the in vitro
translation of these mRNAs examined.

An association between the DBP and the adenovirus DNA polymerase has been
postulated to be important in the precessive nature of replication, but
such an association has never been demonstrated.  We will use synthetic
peptides corresponding to regions of the DBP to determine if this potential
association can be disrupted, and if so, which regions of the DBP are
involved.

The successful outcome of these experiments will provide insight into the
molecular mechanisms by which eukaryotic gene expression is regulated.

## Key facts

- **NIH application ID:** 3127346
- **Project number:** 5R01AI017654-09
- **Recipient organization:** ST. JUDE CHILDREN'S RESEARCH HOSPITAL
- **SAM.gov UEI:** JL4JHE9SDRR3
- **Principal Investigator:** GEOFFREY R KITCHINGMAN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1992
- **Award amount:** $149,573
- **Award type:** 5
- **Project period:** 1980-12-01 → 1994-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3127346

## Citation

> US National Institutes of Health, RePORTER application 3127346, GENETIC AND BIOCHEMICAL ANALYSIS OF AD5 REGION 2 (5R01AI017654-09). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3127346. Licensed CC0.

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