Isolation of the murine interleukin 2 gene and characterization of its regulatory architecture

Interleukin 2 (IL2) is the primary growth hormone used by mature T cells and this lymphokine plays an important role in the magnification of cell-mediated immune responses. Under normal circumstances its expression is limited to antigen-activated type 1 helper T cells (T_H1) and the ability to transcribe this gene is often regarded as evidence for commitment to this developmental lineage. There is, however, abundant evidence than many non-T_H1 T cells, under appropriate conditions, possess the ability to express this gene. Of paramount interest in the study of T-cell development is the mechanisms by which differentiating thymocytes are endowed with particular combinations of cell surface proteins and response repertoires. For example, why do most helper T cells express the CD4 differentiation antigen?

As a first step in understanding these developmental processes the gene encoding IL2 was isolated from a mouse genomic library by probing with a conspecific IL2 cDNA. The sequence of the 5' flanking region from + 1 to -2800 was determined and compared to the previously reported human sequence. Extensive identity exists between +1 and -580 (86%) and sites previously shown to be crucial for the proper expression of the human gene are well conserved in both sequence location in the mouse counterpart.

Transient expression assays were used to evaluate the contribution of various genomic sequences to high-level gene expression mediated by a cloned IL2 promoter fragment. Differing lengths of 5' flanking DNA, all terminating in the 5' untranslated region, were linked to a reporter gene, bacterial chloramphenicol acetyltransferase (CAT) and enzyme activity was measured after introduction into IL2-producing cell lines. No CAT was ever detected without stimulation of the recipient cells. A cloned promoter fragment containing only 321 bp of upstream DNA was expressed well in both Jurkat and EL4.El cells. Addition of intragenic or downstream DNA to these 5' IL2-CAT constructs showed that no obvious regulatory regions resided there. However, increasing the extent of 5' DNA from -321 to -2800 revealed several positive and negative regulatory elements. One negative region that was well characterized resided between -750 and -1000 and consisted almost exclusively of alternating purine and pyrimidines. There is no sequence resembling this in the human gene now, but there is evidence that there may have once been.

No region, when deleted, could relax either the stringent induction-dependence on cell-type specificity displayed by this promoter. Reagents that modulated endogenous IL2 expression, such as cAMP, cyclosporin A, and IL1, affected expression of the 5' IL2-CAT constructs also. For a given reagent, expression from all expressible constructs was suppressed or enhanced to the same extent. This suggests that these modulators affect IL2 expression through perturbation of a central inductive signal rather than by summation of the effects of discrete, independently regulated, negative and positive transcription factors.

Structure-function studies of serine hydrolases: synthesis of a gene for ∝-lytic protease and the purification and characterization of a mutant β-lactamase

The author has constructed a synthetic gene for ∝-lytic protease. Since the DNA sequence of the protein is not known, the gene was designed by using the reverse translation of ∝-lytic protease's amino acid sequence. Unique restriction sites are carefully sought in the degenerate DNA sequence to aid in future mutagenesis studies. The unique restriction sites are designed approximately 50 base pairs apart and their appropriate codons used in the DNA sequence. The codons used to construct the DNA sequence of ∝-lytic protease are preferred codons in E-coli or used in the production of β-lactamase. Codon usage is also distributed evenly to ensure that one particular codon is not heavily used. The gene is essentially constructed from the outside in. The gene is built in a stepwise fashion using plasmids as the vehicles for the ∝-lytic oligomers. The use of plasmids allows the replication and isolation of large quantities of the intermediates during gene synthesis. The ∝-lytic DNA is a double-stranded oligomer that has sufficient overhang and sticky ends to anneal correctly in the vector. After six steps of incorporating ∝-lytic DNA, the gene is completed and sequenced to ensure that the correct DNA sequence is present and that no mutations occurred in the structural gene.

β-lactamase is the other serine hydrolase studied in this thesis. The author used the class A RTEM-1 β- lactamase encoded on the plasmid pBR322 to investigate the roll of the conserved threonine residue at position 71. Cassette mutagenesis was previously used to generate all possible amino acid substitutions at position 71. The work presented here describes the purification and kinetic characterization of a T71H mutant previously constructed by S. Schultz. The mutated gene was transferred into plasmid pJN for expression and induced with IPTG. The enzyme is purified by column chromatography and FPLC to homogeneity. Kinetic studies reveal that the mutant has lower k_(cat) values on benzylpenicillin, cephalothin and 6-aminopenicillanic acid but no changes in k_m except for cephalothin which is approximately 4 times higher. The mutant did not change siginificantly in its pH profile compared to the wild-type enzyme. Also, the mutant is more sensitive to thermal denaturation as compared to the wild-type enzyme. However, experimental evidence indicates that the probable generation of a positive charge at position 71 thermally stabilized the mutant.

Isolation and characterization of proteins that bind to yeast origins of DNA replication

Yeast chromosomes contain sequences called ARSs which function as origins of replication in vitro and in vivo. We have carried out a systematic deletion analysis of ARS1, allowing us to define three functionally distinct domains, designated A, B, and C. Domain A is a sequence of 11 to 19bp, containing the core consensus element that is required for replication. The core consensus sequence, A/TTTTATPuTTTA/T, is conserved at all ARSs sequenced to date. A fragment containing only element A and 8 flanking nucleotides enables autonomous replication of centromeric plasmids. These plasmids replicate very inefficiently, suggesting that flanking sequences must be important for ARS function. Domain B also provides important sequences needed for efficient replication. Deletion of domain B drastically increases the doubling times of transformants and reduces plasmid stability. Domain B contains a potential consensus sequence conserved at some ARSs which overlaps a region of bent DNA. Mutational analysis suggests this bent DNA may be important for ARS function. Deletion of domain C has only a slight effect on replication of plasmids carrying those deletions.

We have identified a protein called ARS binding factor I (ABF-I) that binds to the HMR-E ARS and ARS1. We have purified this protein to homogeneity using conventional and oligonucleotide affinity chromatography. The protein has an apparent molecular weight of 135kDa and is present at about 700 molecules per diploid cell, based on the yield of purified protein and in situ antibody staining. DNaseI footprinting reveals that ABF-I binds sequence-specifically to an approximately 24bp sequence that overlaps element Bat ARSl. This same protein binds to and protects a similar size region at the HMR-E ARS.

We also find evidence for another ARS binding protein, ABF-III, based on DN asei footprint analysis and gel retardation assays. The protein protects approximately 22bp adjacent to the ABF-I site. There appears to be no interaction between ABF-I and ABF-III despite the proximity of their binding sites.

To address the function of ABF-I in DNA replication, we have cloned the ABF-I gene using rabbit polyclonal anti-sera and murine monoclonal antibodies against ABF-I to screen a λgt11 expression library. Four EcoRI restriction fragments were isolated which encoded proteins that were recognized by both polyclonal and monoclonal antibodies. A gene disruption can now be constructed to determine the in vivo function of ABF-I.

Characterization and developmental regulation of a gene expressed specifically in the skeletogenic lineage of the sea urchin embryo

The sea urchin embryonic skeleton, or spicule, is deposited by mesenchymal progeny of four precursor cells, the micromeres, which are determined to the skeletogenic pathway by a process known as cytoplasmic localization. A gene encoding one of the major products of the skeletogenic mesenchyme, a prominent 50 kD protein of the spicule matrix, has been characterized in detail. cDNA clones were first isolated by antibody screening of a phage expression library, followed by isolation of homologous genomic clones. The gene, known as SM50, is single copy in the sea urchin genome, is divided into two exons of 213 and 1682 bp, and is expressed only in skeletogenic cells. Transcripts are first detectable at the 120 cell stage, shortly after the segregation of the skeletogenic precursors from the rest of the embryo. The SM50 open reading frame begins within the first exon, is 450 amino acids in length, and contains a loosely repeated 13 amino acid motif rich in acidic residues which accounts for 45% of the protein and which is possibly involved in interaction with the mineral phase of the spicule.

The important cis-acting regions of the SM50 gene necessary for proper regulation of expression were identified by gene transfer experiments. A 562 bp promoter fragment, containing 438 bp of 5' promoter sequence and 124 bp of the SM50 first exon (including the SM50 initiation codon), was both necessary and sufficient to direct high levels of expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene specifically in the skeletogenic cells. Removal of promoter sequences between positions -2200 and -438, and of transcribed regions downstream of +124 (including the SM50 intron), had no effect on the spatial or transcriptional activity of the transgenes.

Regulatory proteins that interact with the SM50 promoter were identified by the gel retardation assay, using bulk embryo mesenchyme blastula stage nuclear proteins. Five protein binding sites were identified and mapped to various degrees of resolution. Two sites are homologous, may be enhancer elements, and at least one is required for expression. Two additional sites are also present in the promoter of the aboral ectoderm specific cytoskeletal actin gene CyIIIa; one of these is a CCAA T element, the other a putative repressor element. The fifth site overlaps the binding site of the putative repressor and may function as a positive regulator by interfering with binding of the repressor. All of the proteins are detectable in nuclear extracts prepared from 64 cell stage embryos, a stage just before expression of SM50 is initiated, as well as from blastula and gastrula stage; the putative enhancer binding protein may be maternal as well.

Engineered underdominance as a method of insect population replacement and reproductive isolation

Insect vector-borne diseases, such as malaria and dengue fever (both spread by mosquito vectors), continue to significantly impact health worldwide, despite the efforts put forth to eradicate them. Suppression strategies utilizing genetically modified disease-refractory insects have surfaced as an attractive means of disease control, and progress has been made on engineering disease-resistant insect vectors. However, laboratory-engineered disease refractory genes would probably not spread in the wild, and would most likely need to be linked to a gene drive system in order to proliferate in native insect populations. Underdominant systems like translocations and engineered underdominance have been proposed as potential mechanisms for spreading disease refractory genes. Not only do these threshold-dependent systems have certain advantages over other potential gene drive mechanisms, such as localization of gene drive and removability, extreme engineered underdominance can also be used to bring about reproductive isolation, which may be of interest in controlling the spread of GMO crops. Proof-of-principle establishment of such drive mechanisms in a well-understood and studied insect, such as Drosophila melanogaster, is essential before more applied systems can be developed for the less characterized vector species of interest, such as mosquitoes. This work details the development of several distinct types of engineered underdominance and of translocations in Drosophila, including ones capable of bringing about reproductive isolation and population replacement, as a proof of concept study that can inform efforts to construct such systems in insect disease vectors.