Gene enrichment using antibodies to DNA/RNA hybrids : mapping the ribosomal DNA of slime mold and rat

A novel method for gene enrichment has been developed and applied to mapping the rRNA genes of two eucaryotic organisms. The method makes use of antibodies to DNA/RNA hybrids prepared by injecting rabbits with the synthetic hybrid poly(rA)•poly(dT). Antibodies which cross-react with non-hybrid nucleic acids were removed from the purified IgG fraction by adsorption on columns of DNA-Sepharose, oligo(dT)-cellulose, and poly(rA)-Sepharose. Subsequent purification of the specific DNA/RNA hybrid antibody was carried out on a column of oligo(dT)-cellulose to which poly(rA) was hybridized. Attachment of these antibodies to CNBr-activated Sepharose produced an affinity resin which specifically binds DNA/RNA hybrids.

In order to map the rDNA of the slime mold Dictyostelium discoideum, R-loops were formed using unsheared nuclear DNA and the 178 and 268 rRNAs of this organism. This mixture was passed through a column containing the affinity resin, and bound molecules containing R- loops were eluted by high salt. This purified rDN A was observed directly in the electron microscope. Evidence was obtained that there is a physical end to Dictyostelium rDN A molecules approximately 10 kilobase pairs (kbp) from the region which codes for the 268 rRNA. This finding is consistent with reports of other investigators that the rRNA genes exist as inverse repeats on extra-chromosomal molecules of DNA unattached to the remainder of the nuclear DNA in this organism.

The same general procedure was used to map the rRNA genes of the rat. Molecules of DNA which contained R-loops formed with the 188 and 288 rRNAs were enriched approximately 150- fold from total genomal rat DNA by two cycles of purification on the affinity column. Electron microscopic measurements of these molecules enabled the construction of an R-loop map of rat rDNA. Eleven of the observed molecules contained three or four R-loops or else two R-loops separated by a long spacer. These observations indicated that the rat rRNA genes are arranged as tandem repeats. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. These eleven molecules may represent repeating units of exactly the same length within the errors of the measurements, although a certain degree of length heterogeneity cannot be ruled out. If significantly shorter or longer repeating units exist, they are probably much less common than the 37.2 kbp unit.

The last section of the thesis describes the production of antibodies to non-histone chromosomal proteins which have been exposed to the ionic detergent sodium dodecyl sulfate (SDS). The presence of low concentrations of SDS did not seem to affect either production of antibodies or their general specificity. Also, a technique is described for the in situ immunofluorescent detection of protein antigens in polyacrylamide gels.

Distinct intron DNA structures in simian virus 40 T-antigen and adenovirus 2 E1A genes

Distinct structures delineating the introns of Simian Virus 40 T-antigen and Adenovirus 2 E1A genes have been discovered. The structures, which are centered around the branch points of the genes inserted in supercoiled double-stranded plasmids, are specifically targeted through photoactivated strand cleavage by the metal complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III). The DNA sites that are recognized lack sequence homology but are similar in demarcating functionally important sites on the RNA level. The single-stranded DNA fragments corresponding to the coding strands of the genes were also found to fold into a structure apparently identical to that in the supercoiled genes based on the recognition by the metal complex. Further investigation of different single-stranded DNA fragments with other structural probes, such as another metal complex bis(1,10-phenanthroline)(phenanthrenequinone diimine)rhodium(III), AMT (4'aminomethyl-4,5',8 trimethylpsoralen), restriction enzyme Mse I, and mung bean nuclease, showed that the structures require the sequ ences at both ends of the intron plus the flanking sequences but not the middle of the intron. The two ends form independent helices which interact with each other to form the global tertiary structures. Both of the intron structures share similarities to the structure of the Holliday junction, which is also known to be specifically targeted by the former metal complex. These structures may have arisen from early RNA intron structures and may have been used to facilitate the evolution of genes through exon shuffling by acting as target sites for recombinase enzymes.

In vivo analysis of interactions between trans-acting factors and their target genes

The investigations presented in this thesis use various in vivo techniques to understand how trans-acting factors control gene expression. The first part addresses the transcriptional regulation of muscle creatine kinase (MCK). MCK expression is activated during the course of development and is found only in differentiated muscle. Several in vivo footprints are observed at the enhancer of this gene, but all of these interactions are limited to cell types that express MCK. This is interesting because two of the footprints appear to represent muscle specific use of general transcription factors, while the other two correspond to sites that can bind the myogenic regulator, MyoD1, in vitro. MyoD1 and these general factors are present in myoblasts, but can bind to the enhancer only in myocytes. This suggests that either the factors themselves are post-translationally modified (phosphorylation or protein:protein interactions), or the accessibility of the enhancer to the factors is limited (changes in chromatin structure). The in vivo footprinting study of MCK was performed with a new ligation mediated, single-sided PCR (polymerase chain reaction) technique that I have developed.

The second half of the thesis concerns the regulation of mouse metallothionein (MT). Metallothioneins are a family of highly conserved housekeeping genes whose expression can be induced by heavy metals, steroids, and other stresses. By adapting a primer extension method of genomic sequencing to in vivo footprinting, I've observed both metal inducible and noninducible interactions at the promoter of MT-I. From these results I've been able to limit the possible mechanisms by which metal responsive trans-acting factors induce transcription. These interpretations correlate with a second line of experiments involving the stable titration of positive acting factors necessary for induction of MT. I've amplified the promoter of MT to 10^2-10^3 copies per cell by fusing the 5' and 3' ends of the MT gene to the coding region of DHFR and selecting cells for methotrexate resistance. In these cells, there is a metal-specific titration effect, and although it acts at the level of transcription, it appears to be independent of direct DNA binding factors.

Studies of bacteriophage T4 tail fibers and tail fiber genes

The distal half of the bacteriophage T4 tail fiber interacts with the surface of the bacterium during adsorption. The largest polypeptide in this half fiber is the product of gene 37 (P37). During assembly of the tail fiber, P37 interacts with the product of gene 38 (P38). These two gene products are incompatible with the corresponding gene products from the related phage T2. T2 P37 does not interact with T4 P38 and T2 P38 does not interact with T4 P37. Crosses between T2 and T4 phages mutant in genes 37 and 38 have shown that the carboxyl end of P37 interacts with P38 and with the bacterial surface. In the corresponding region of gene 37 and in gene 38 there is no recombination between T2 and T4. In the rest of gene 37 there are two small regions with relatively high recombination and a region of low recombination.

When T2/T4 heteroduplex DNA molecules are examined in the electron microscope four nonhomologous loops appear in the region of genes 37 and 38. Heteroduplexes between hybrid phages which have part of gene 37 from T4 and part from T2 have roughly located gene 37 mutations in the heteroduplex pattern. For a more precise location of the , mutations a physical map of gene 37 was constructed by determining the molecular weights of amber polypeptide fragments on polyacrylamide gels in the presence of sodium dodecyl sulfate. When the physical and heteroduplex maps are aligned, the regions of low recombination correspond to regions of nonhomology between T2 and T4. Regions with relatively high recombination are homologous.

The molecular weight of T2 P37 is about 13,000 greater than that of T4 P37. Analysis of hybrid phage has shown that this molecular weight difference is all at the carboxyl end of P37.

An antiserum has been prepared which is specific for the distal half fiber of T4. Tests of the ability of gene 37 hybrids to block this antiserum show that there are at least 4 subclasses of antigen specified by different parts of P37.

Observations in the electron microscope of the tailfiber - anti- body complexes formed by the gene 37 hybrids and the specific anti- serum have shown that P37 is oriented linearly in the distal half fiber with its N-terminus near the joint between the two half fibers and its C-terminus near the tip of the fiber. These observations lead to a simple model for the structure of the distal half fiber.

The high recombination in T4 gene 34 was also investigated. A comparison of genetic and physical maps of gene 34 showed that there is a gradient of increasing recombination near one end of the gene.