A NEW TUMOR SUPPRESSOR GENE CANDIDATE REGULATED BY THE NON-CODING RNA PCA3 IN HUMAN PROSTATE CANCER

Prostate cancer is the second leading cause of cancer-related death and the most common non-skin cancer in men in the USA. Considerable advancements in the practice of medicine have allowed a significant improvement in the diagnosis and treatment of this disease and, in recent years, both incidence and mortality rates have been slightly declining. However, it is still estimated that 1 man in 6 will be diagnosed with prostate cancer during his lifetime, and 1 man in 35 will die of the disease. In order to identify novel strategies and effective therapeutic approaches in the fight against prostate cancer, it is imperative to improve our understanding of its complex biology since many aspects of prostate cancer initiation and progression still remain elusive. The study of tumor biomarkers, due to their specific altered expression in tumor versus normal tissue, is a valid tool for elucidating key aspects of cancer biology, and may provide important insights into the molecular mechanisms underlining the tumorigenesis process of prostate cancer. PCA3, is considered the most specific prostate cancer biomarker, however its biological role, until now, remained unknown. PCA3 is a long non-coding RNA (ncRNA) expressed from chromosome 9q21 and its study led us to the discovery of a novel human gene, PC-TSGC, transcribed from the opposite strand and in an antisense orientation to PCA3. With the work presented in this thesis, we demonstrate that PCA3 exerts a negative regulatory role over PC-TSGC, and we propose PC-TSGC to be a new tumor suppressor gene that contrasts the transformation of prostate cells by inhibiting Rho-GTPases signaling pathways. Our findings provide a biological role for PCA3 in prostate cancer and suggest a new mechanism of tumor suppressor gene inactivation mediated by non-coding RNA. Also, the characterization of PCA3 and PC-TSGC led us to propose a new molecular pathway involving both genes in the transformation process of the prostate, thus providing a new piece of the jigsaw puzzle representing the complex biology of prostate cancer.

Post-transcriptional Regulation of Mammalian Gene Expression in Non-coding Region of Target RNA

Tumor Suppressor Candidate 2 (TUSC2) is a novel tumor suppressor gene located in the human chromosome 3p21.3 region. TUSC2 mRNA transcripts could be detected on Northern blots in both normal lung and some lung cancer cell lines, but no endogenous TUSC2 protein could be detected in a majority of lung cancer cell lines. Mechanisms regulating TUSC2 protein expression and its inactivation in primary lung cancer cells are largely unknown. We investigated the role of the 5’- and 3’-untranslated regions (UTRs) of the TUSC2 gene in the regulation of TUSC2 protein expression. We found that two small upstream open-reading frames (uORFs) in the 5’UTR of TUSC2 could markedly inhibit the translational initiation of TUSC2 protein by interfering with the “scanning” of the ribosome initiation complexes. Site-specific stem-loop array reverse transcription-polymerase chain reaction (SLA-RT-PCR) verified several micoRNAs (miRNAs) targeted at 3’UTR and directed TUSC2 cleavage and degradation. In addition, we used the established let-7-targeted high mobility group A2 (Hmga2) mRNA as a model system to study the mechanism of regulation of target mRNA by miRNAs in mammalian cells under physiological conditions. There have been no evidence of direct link between mRNA downregulation and mRNA cleavages mediated by miRNAs. Here we showed that the endonucleolytic cleavages on mRNAs were initiated by mammalian miRNA in seed pairing style. Let-7 directed cleavage activities among the eight predicted potential target sites have varied efficiency, which are influenced by the positional and the structural contexts in the UTR. The 5’ cleaved RNA fragments were mostly oligouridylated at their 3’-termini and accumulated for delayed 5’–3’ degradation. RNA fragment oligouridylation played important roles in marking RNA fragments for delayed bulk degradation and in converting RNA degradation mode from 3’–5’ to 5’–3’ with cooperative efforts from both endonucleolytic and non-catalytic miRNA-induced silencing complex (miRISC). Our findings point to a mammalian miRNA-mediated mechanism for the regulation of mRNA that miRNA can decrease target mRNA through target mRNA cleavage and uridine addition

Systems Biology Approaches to Probe Gene Regulation in Bacteria

Mechanisms that allow pathogens to colonize the host are not the product of isolated genes, but instead emerge from the concerted operation of regulatory networks. Therefore, identifying components and the systemic behavior of networks is necessary to a better understanding of gene regulation and pathogenesis. To this end, I have developed systems biology approaches to study transcriptional and post-transcriptional gene regulation in bacteria, with an emphasis in the human pathogen Mycobacterium tuberculosis (Mtb). First, I developed a network response method to identify parts of the Mtb global transcriptional regulatory network utilized by the pathogen to counteract phagosomal stresses and survive within resting macrophages. As a result, the method unveiled transcriptional regulators and associated regulons utilized by Mtb to establish a successful infection of macrophages throughout the first 14 days of infection. Additionally, this network-based analysis identified the production of Fe-S proteins coupled to lipid metabolism through the alkane hydroxylase complex as a possible strategy employed by Mtb to survive in the host. Second, I developed a network inference method to infer the small non-coding RNA (sRNA) regulatory network in Mtb. The method identifies sRNA-mRNA interactions by integrating a priori knowledge of possible binding sites with structure-driven identification of binding sites. The reconstructed network was useful to predict functional roles for the multitude of sRNAs recently discovered in the pathogen, being that several sRNAs were postulated to be involved in virulence-related processes. Finally, I applied a combined experimental and computational approach to study post-transcriptional repression mediated by small non-coding RNAs in bacteria. Specifically, a probabilistic ranking methodology termed rank-conciliation was developed to infer sRNA-mRNA interactions based on multiple types of data. The method was shown to improve target prediction in Escherichia coli, and therefore is useful to prioritize candidate targets for experimental validation.

A TEMPERATURE-SENSITIVE DEFECT IN THE EXPRESSION OF A SARCOMA VIRUS TRANSFORMING GENE

The viral proteins synthesized by a Moloney murine sarcoma virus (Mo-MuSV) with a temperature-sensitive mutation in a function required for the maintenance of the transformed state (ts110) were examined. Normal rat kidney cells (NRK) were infected with the ts110 virus and a non-virus-producing cell clone, termed 6m2, was isolated. This cell clone had a malignant phenotype at 33(DEGREES), the permissive temperature, but changed to a normal phenotype at 39(DEGREES).^ Two viral proteins were detected in 6m2 cells. A 58,000 dalton protein (P58) was detected at both 33(DEGREES) and 39(DEGREES) and contained only core protein (gag) coded sequences. An 85,000 dalton protein (P85) was detected only at 33(DEGREES) and contained sequences of viral core proteins p15, pp12, and part of p30 as well as protein sequences attributed by peptide mapping to P23 and P38, two candidate viral mouse src (v-mos) gene products. These results provide good evidence that P85 is a gag-mos polyprotein. As expected for a functional mos-gene product, P85 synthesis preceded parameters characteristic of the transformed state, including changes in cell morphology, in the cytoplasmic microtubule complex (CMTC) and in the rate of hexose uptake.^ Other studies were conducted to ascertain the defect which prohibited the synthesis of P85 at 39(DEGREES), the non-permissive temperature. When 6m2 cells were treated with actinomycin D at 39(DEGREES) and shifted to 33(DEGREES), the cells were unable to synthesize P85, but P58 continued to be made. P85 mRNA, active at 33(DEGREES), continued to be translated for two to three hours after shifting to 39(DEGREES) as judged by pulse-labeling experiments. Virus harvested at 33(DEGREES) from ts110 MuSV producer cells packaged both P85 and P58 coding RNAs while virus harvested at 39(DEGREES) was deficient in the amount of P85 coding RNA. Agarose gel electrophoresis of 6m2 cellular RNA showed that RNA harvested at 33(DEGREES) contained the 4.0 and 3.5 kb RNAs. Similar experiments on cells maintained at 39(DEGREES) have detected only the 4.0 kb RNA, suggesting that the 3.5 kb RNA codes for P85. The defect appeared to be in the long term stability of the P85 coding RNA at 39(DEGREES), since, in shift-up experiments (33(DEGREES) (--->) 39(DEGREES)), P85 was translated for only three hours at 39(DEGREES), while P58 was synthesized for at least eight hours. However, at 33(DEGREES) in the presence of actinomycin D, the ratio of P85 and P58 synthesis at hourly intervals was similar throughout a 12 hour period. ^

RNA binding characteristics and overall topology of the vaccinia virus polyadenylation complex

mRNA 3′ polyadenylation is central to mRNA biogenesis in prokaryotes and eukaryotes, and is implicated in numerous aspects of mRNA metabolism, including efficiency of mRNA export from the nucleus, message stability, and initiation of translation. However, due to the great complexity of the eukaryotic polyadenylation apparatus, the mechanisms of RNA 3 ′ end processing have remained elusive. Although the RNA processing reactions leading to polyadenylated messenger RNA have been studied in many systems, and much progress has been made, a complete understanding of the biochemistry of the poly(A) polymerase enzyme is still lacking. My research uses Vaccinia virus as a model system to gain a better understanding of this complicated polyadenylation process, which consist of RNA binding, catalysis and polymerase translocation. ^ Vaccinia virus replicates in the cytoplasm of its host cell, so it must employ its own poly(A) polymerase (PAP), a heterodimer of two virus encoded proteins, VP55 and VP39. VP55 is the catalytic subunit, adding 30 adenylates to a non-polyadenylated RNA in a rapid processive manner before abruptly changing to a slow, non-processive mode of adenylate addition and dissociating from the RNA. VP39 is the stimulatory subunit. It has no polyadenylation catalytic activity by itself, but when associated with VP55 it facilitates the semi-processive synthesis of tails several hundred adenylates in length. ^ Oligonucleotide selection and competition studies have shown that the heterodimer binds a minimal motif of (rU)2 (N)25 U, the “heterodimer binding motif”, within an oligonucleotide, and its primer selection for polyadenylation is base-type specific. ^ Crosslinking studies using photosensitive uridylate analogs show that within a VP55-VP39-primer ternary complex, VP55 comes into contact with all three required uridylates, while VP39 only contacts the downstream uridylate. Further studies, using a backbone-anchored photosensitive crosslinker show that both PAP subunits are in close proximity to the downstream −10 to −21 region of 50mer model primers containing the heterodimer binding motif. This equal crosslinking to both subunits suggests that the dimerization of VP55 and VP39 creates either a cleft or a channel between the two subunits through which this region of RNA passes. ^ Peptide mapping studies of VP39 covalently crosslinked to the oligonucleotide have identified residue R107 as the amino acid in close proximity to the −10 uridylate. This helps us project a conceptual model onto the known physical surface of this subunit. In the absence of any tertiary structural data for VP55, we have used a series of oligonucleotide selection assays, as well as crosslinking, nucleotide transfer assays, and gel shift assays to gain insight into the requirements for binding, polyadenylation and translocation. Collectively, these data allow us to put together a comprehensive model of the structure and function of the polyadenylation ternary complex consisting of VP39, VP55 and RNA. ^

Regulation of two P450 isoforms: CYP2D6 and CYP4F11

Cytochromes P450 catalyze a monooxygenase reaction in which molecular oxygen is split and one oxygen atom is incorporated into the substrate. As a whole, P450 researchers have focused most of their attention on substrate metabolism and relatively little on how these enzymes are regulated. This study will focus on the regulation of two P450 isoforms known as, CYP2D6 and CYP4F11. ^ The human CYP2D gene locus contains two pseudogenes and one functional gene known as CYP2D6. This locus is highly polymorphic and produces several alternatively spliced transcripts from the pseudogene CYP2D7. My objective was to understand the role of SV5-in (splice variant 5), one of several alternative splice variants transcribed from the CYP2D7 pseudogene. My results indicate that SV5-in mRNA causes an increase in CYP2D6 protein levels and suggest that there is a role for SV5-in in regulation of CYP2D6 expression. ^ Second, CYP4F11 is a recently discovered and uncharacterized isoform, derived from the CYP4F subfamily. It metabolizes several clinically relevant drugs (i.e.—erythromycin and benzphetamine) and some endogenous inflammatory mediators (i.e.—LTB4). After evaluation of microarray data, I observed an increase in CYP4F11 mRNA levels from wild-type HCT116 cells compared to p53-null cells. Our objectives were to explore and understand this connection between p53 and CYP4F11. Microarray data were confirmed by Q-PCR, after which this effect was again observed at the protein level via Western blot and again at the promoter level via luciferase assay and chromatin immunoprecipitation. Our results indicate that p53 protein regulates expression of CYP4F11 mRNA and protein through CYP4F11 promoter binding (note that p53 binding to CYP4F11 DNA was not shown to be direct). These results signify a whole new level of regulation of drug metabolizing enzymes by p53. ^ An understanding of CYP4F11 regulation by p53 could help us understand another pathway leading to apoptosis or cell growth arrest. This can aid future drug studies and discover new drug metabolism pathways under the control of a tumor suppressor protein. An understanding of the CYP2D6 regulation pathway could illuminate the role of non-coding RNAs in the P450 field and potentially explain several inter-individual drug response variations observed in clinical medicine that are not yet completely explained by genotyping analysis. ^

Association of folate metabolic pathway genes with human spina bifida meningomyelocele

Neural tube defects including spina bifida meningomyelocele (SBMM) are common malformations of the brain and spinal cord, and include all abnormalities resulting from lack of closure of the developing neural tube during embryological development.^ The specific aims of this study were to determine if single nucleotide polymorphic variants (SNPs) in the folate/homocysteine metabolic pathway genes confer a risk for NTD susceptibility within this SBMM population.^ In completion of the first specific aim, two novel SNPs were identified in the FOLR1 gene in Chromosome 11of patients including one in non-coding exon 1 with a C → T transition at nucleotide position 71578317 and another in non-coding exon 3 with a T → G transversion at nucleotide position 71579123. It will be important to determine if these variants are present in the respective parents of these individuals. If they are in fact de novo variants, then these SNPs may be more likely to contribute to the birth defect.^ The second project aim was to analyze genotypes associated with SBMM risk by transmission disequilibrium tests (TDT) and association was detected on several SNPs across the folate metabolic pathway genes in this population. SNPs with significant RC-TDT values were found within the DHFR gene (rs1650723), the MTRR gene (rs327592), the FOLR2 gene (rs13908), four tightly linked variants in the FOLR3 gene (rs7925545, rs7926875, rs7926987, rs7926360) and a variant in the SLC19A1 gene (rs1888530). The product of each of these genes performs a vital function in the folate metabolic pathway. It is conceivable, therefore, that if the individual SNP or SNPs can be proven to perturb the function in some way that they may be involved in the disruption of folate metabolism and in the resulting birth defect. Validating the results of this study in other independent populations will further strengthen the evidence that dysfunction of folate enzymes and receptors may confer SBMM risk in humans. ^

A molecular analysis of the conditional defect in MuSVts 110 viral RNA splicing

Cells infected with MuSVts110 express a viral RNA which contains an inherent conditional defect in RNA splicing. It has been shown previously that splicing of the MuSVts110 primary transcript is essential to morphological transformation of 6m2 cells in vitro. A growth temperature of 33$\sp\circ$C is permissive for viral RNA splicing,and, consequently, 6m2 cells appear morphologically transformed at this temperature. However, 6m2 cells appear phenotypically normal when incubated at 39$\sp\circ$C, the non-permissive temperature for viral RNA splicing.^ After a shift from 39$\sp\circ$C to 33$\sp\circ$C, the coordinate splicing of previously synthesized and newly transcribed MuSVts110 RNA was achieved. By S1 nuclease analysis of total RNA isolated at various times, 5$\sp\prime$ splice site cleavage of the MuSVts110 transcript appeared to occur 60 minutes after the shift to 33$\sp\circ$C, and 30 minutes prior to detectable exon ligation. In addition, consistent with the permissive temperatures and the kinetic timeframe of viral RNA splicing after a shift to 33$\sp\circ$C, four temperature sensitive blockades to primer extension were identified 26-75 bases upstream of the 3$\sp\prime$ splice site. These blockades likely reflect four branchpoint sequences utilized in the formation of MuSVts110 lariat splicing-intermediates.^ The 54-5A4 cell line is a spontaneous revertant of 6m2 cells and appears transformed at all growth temperatures. Primer extension sequence analysis has shown that a five base deletion occurred at the 3$\sp\prime$ splice site in MuSVts110 RNA allowing the expression of a viral transforming protein in 54-5A4 in the absence of RNA splicing, whereas in the parental 6m2 cell line, a splicing event is necessary to generate a similar transforming protein. As a consequence of this deletion, splicing cannot occur and the formation of the four MuSVts110 branched-intermediates were not observed at any temperature in 54-5A4 cells. However, 5$\sp\prime$ splice site cleavage was still detected at 33$\sp\circ$C.^ Finally, we have investigated the role of the 1488 bp deletion which occurred in the generation of MuSVts110 in the activation of temperature sensitive viral RNA splicing. This deletion appears solely responsible for splice site activation. Whether intron size is the crucial factor in MuSVts110 RNA splicing or whether inhibitory sequences were removed by the deletion is currently unknown. (Abstract shortened with permission of author.) ^

Exon involvement in the regulation of MuSVts110 RNA splicing

MuSVts110 is a conditionally defective mutant of Moloney murine sarcoma virus which undergoes a novel tmperature-dependent splice event at growth temperatures of 33$\sp\circ$C or lower. Relative to wild-type MuSV-124, MuSVts110 contains a 1487 base deletion spanning from the 3$\sp\prime$ end of the p30 gag coding region to just downstream of the first v-mos initiation codon. As a result, the gag and mos genes are fused out of frame and no v-mos protein is expressed. However, upon a shift to 33$\sp\circ$C or lower, a splice event occurs which removes 431 bases, realigns the gag and mos genes, and allows read-through translation of a P85gag-mos transforming protein. Interestingly, while the cryptic splice sites utilized in MuSVts110 are present and unaltered in MuSV-124, they are never used. Due to the 1487 base deletion, the MuSV-124 intron was reduced from 1919 to 431 bases suggesting that intron size might be involved in the activation of these cryptic splice sites in MuSVts110. Since the splicing phenotype of the MuSVts110 equivalent (TS32 DNA) which contains the identical 1487 base deletion introduced into otherwise wild-type MuSV-124 DNA, was indistinguishable from authentic MuSVts110, it was concluded that this deletion alone is responsible for activation of the cryptic splice sites used in MuSVts110. These results also confirmed that thermodependent splicing is an intrinsic property of the viral RNA and not due to some cellular defect. Furthermore, analysis of gag gene deletion and frameshift MuSVts110 mutants demonstrated that viral gag gene proteins do not play a role in regulation of MuSVts110 splicing. Instead, cis-acting viral sequences appear to mediate regulation of the splice event.^ Our initial observation that truncation of the MuSVts110 transcript, leaving only residual amounts of the flanking exon sequences, completely abolished splicing activity argued that exon sequences might participate in the regulation of the splice event.^ Analysis of exon sequence involvement has also identified cis-acting sequences important in the thermodependence of the splice event. Data suggest that regulation of the MuSVts110 splice event involves multiple interactions between specific intron and exon sequences and spliceosome components which together limit splicing activity to temperatures of 33$\sp\circ$C or lower while simultaneously restricting splicing to a maximum of 50% efficiency. (Abstract shortened with permission of author.) ^

c-{\it mos\/} RNA expression in somatic cells and its physiological significance in cell cycle progression

The c-mos proto-oncogene, which is expressed at relatively high levels in male and female germ cells, plays a key role in oocyte meiotic maturation. The c-mos gene product in oocytes (p39$\sp{\rm c-mos}$) is necessary and sufficient to initiate meiosis. p39$\sp{\rm c-mos}$ is also an essential component of the cytostatic factor, which is responsible for arresting vertebrate oocytes at the second meiotic metaphase by stabilizing the maturation promoting factor (MPF). MPF is a universal regulator of both meiosis and mitosis. Much less is understood about c-mos expression and function in somatic cells. In addition to gonadal tissues, c-Mos has been detected in some somatic tissues and non-germ cell lines including NIH 3T3 cells as a protein termed p43$\sp{\rm c-mos}$. Since c-mos RNA transcripts were not previously detected in this cell line by Northern blot or S1 protection analyses, a search was made for c-mos RNA in NIH 3T3 cells. c-mos transcripts were detected using the highly sensitive RNA-PCR method and RNase protection assays. Furthermore, cell cycle analyses indicated that expression of c-mos RNA is tightly controlled in a cell cycle dependent manner with highest levels of transcripts (approximately 5 copies/cell) during the G2 phase.^ In order to determine the physiological significance of c-mos RNA expression in somatic cells, antisense mos was placed under the control of an inducible promoter and introduced into either NIH 3T3 cells or C2 cells. It was found that a basal level of expression of antisense mos resulted in interference with mitotic progression and growth arrest. Several nuclear abnormalities were observed, especially the appearance of binucleated and multinucleated cells as well as the extrusion of microvesicles containing cellular material. These results indicate that antisense mos expression results in a block in cytokinesis. In summary, these results establish that c-mos expression is not restricted to germ cells, but instead indicate that c-mos RNA expression occurs during the G2 stage of the cell cycle. Furthermore, these studies demonstrate that the c-mos proto-oncogene plays an important role in cell cycle progression. As in meiosis, c-mos may have a similar but not identical function in regulating cell cycle events in somatic cells, particularly in controlling mitotic progression via activation/stabilization of MPF. ^

Intron involvement in the regulation of MuSVts110 RNA splicing

The murine sarcoma virus MuSVts110 exhibits an alternative RNA splicing pattern. Like other simple retroviruses, MuSVts110 pre-mRNA splicing is balanced to allow the production of both spliced and unspliced RNA during the replicative cycle. In addition to balance, MuSVts110 RNA splicing exhibits a unique growth-temperature restriction to splicing; temperatures below 33$\sp\circ$C are permissive for splicing while temperatures of 37$\sp\circ$C or above are non-permissive. Previous work has established that this thermosensitive splicing phenotype is mediated in cis by viral transcript features. Here we show that at least three sequence elements regulate the MuSVts110 splicing phenotype. First, the MuSVts110 branchpoint (BP) and poly-pyrimidine tract (PPT) were found to be determinants of overall splicing efficiency. Wild-type MuSVts110 possesses a weak BP and PPT adjacent to the 3$\sp\prime$ splice site. Introduction of a strong BP caused MuSVts110 splicing to proceed to virtual completion in vivo, thus losing any vestige of balance or thermosensitivity. In in vitro splicing extracts, the strong BP overcame a blockade to wt MuSVts110 splicing at both the first and second catalytic steps. Weakening the consensus nature of the strong BP allowed the recovery of thermosensitive splicing in vivo, and reinstated the blockades to splicing in vitro, arguing that a suboptimal BP is an unusual manifestation of the proportional splicing pattern of retroviruses. The PPT is essential for accurate recognition of the BP sequence by the splicing machinery. Lengthening the PPT of MuSVts110 from 9 to 19 consecutive pyrimidines increased the overall efficiency of splicing in vivo dramatically, but was less effective than the strong BP in overriding the restriction on splicing imposed by high growth temperatures. Finally, decreasing gradually the overall size of the intron unexpectedly reduced splicing efficiency at growth temperatures permissive for splicing, suggesting that non-conserved sequences within the intron of MuSVts110 participate in splicing regulation as well. Taken together, these results suggest a mechanism of control in which MuSVts110 splicing is modulated by the entire intron, but principally by suboptimal signals at the splice acceptor site. Furthermore, this retroviral system provides a powerful genetic method for selection and analysis of mutations that affect splicing. ^

Involvement of a region of 23S ribosomal RNA of {\it E. coli\/} in decoding of termination codons

Involvement of E. coli 23S ribosomal RNA (rRNA) in decoding of termination codons was first indicated by the characterization of a 23S rRNA mutant that causes UGA-specific nonsense suppression. The work described here was begun to test the hypothesis that more 23S rRNA suppressors of specific nonsense mutations can be isolated and that they would occur non-randomly in the rRNA genes and be clustered in specific, functionally significant regions of rRNA.^ Approximately 2 kilobases of the gene for 23S rRNA were subjected to PCR random mutagenesis and the amplified products screened for suppression of nonsense mutations in trpA. All of the suppressor mutations obtained were located in a thirty-nucleotide part of the GTPase center, a conserved rRNA sequence and structure, and they and others made in that region by site-directed mutagenesis were shown to be UGA-specific in their suppression of termination codon mutations. These results proved the initial hypothesis and demonstrated that a group of nucleotides in this region are involved in decoding of the UGA termination codon. Further, it was shown that limitation of cellular availability or synthesis of L11, a ribosomal protein that binds to the GTPase center rRNA, resulted in suppression of termination codon mutations, suggesting the direct involvement of L11 in termination in vivo.^ Finally, in vivo analysis of certain site-specific mutations made in the GTPase center RNA demonstrated that (a) the G$\cdot$A base pair closing the hexanucleotide hairpin loop was not essential for normal termination, (b) the "U-turn" structure in the 1093 to 1098 hexaloop is critical for normal termination, (c) nucleotides A1095 and A1067, necessary for the binding to ribosomes of thiostrepton, an antibiotic that inhibits polypeptide release factor binding to ribosomes in vitro, are also necessary for normal peptide chain termination in vivo, and (d) involvement of this region of rRNA in termination is determined by some unique subset structure that includes particular nucleotides rather than merely by a general structural feature of the GTPase center.^ This work advances the understanding of peptide chain termination by demonstrating that the GTPase region of 23S rRNA participates in recognition of termination codons, through an associated ribosomal protein and specific conserved nucleotides and structural motifs in its RNA. ^

Functions and intramolecular interactions of ribosomal RNA in decoding of termination codons

Two regions in the 3$\prime$ domain of 16S rRNA (the RNA of the small ribosomal subunit) have been implicated in decoding of termination codons. Using segment-directed PCR random mutagenesis, I isolated 33 translational suppressor mutations in the 3$\prime$ domain of 16S rRNA. Characterization of the mutations by both genetic and biochemical methods indicated that some of the mutations are defective in UGA-specific peptide chain termination and that others may be defective in peptide chain termination at all termination codons. The studies of the mutations at an internal loop in the non-conserved region of helix 44 also indicated that this structure, in a non-conserved region of 16S rRNA, is involved in both peptide chain termination and assembly of 16S rRNA.^ With a suppressible trpA UAG nonsense mutation, a spontaneously arising translational suppressor mutation was isolated in the rrnB operon cloned into a pBR322-derived plasmid. The mutation caused suppression of UAG at two codon positions in trpA but did not suppress UAA or UGA mutations at the same trpA positions. The specificity of the rRNA suppressor mutation suggests that it may cause a defect in UAG-specific peptide chain termination. The mutation is a single nucleotide deletion (G2484$\Delta$) in helix 89 of 23S rRNA (the large RNA of the large ribosomal subunit). The result indicates a functional interaction between two regions of 23S rRNA. Furthermore, it provides suggestive in vivo evidence for the involvement of the peptidyl-transferase center of 23S rRNA in peptide chain termination. The $\Delta$2484 and A1093/$\Delta$2484 (double) mutations were also observed to alter the decoding specificity of the suppressor tRNA lysT(U70), which has a mutation in its acceptor stem. That result suggests that there is an interaction between the stem-loop region of helix 89 of 23S rRNA and the acceptor stem of tRNA during decoding and that the interaction is important for the decoding specificity of tRNA.^ Using gene manipulation procedures, I have constructed a new expression vector to express and purify the cellular protein factors required for a recently developed, realistic in vitro termination assay. The gene for each protein was cloned into the newly constructed vector in such a way that expression yielded a protein with an N-terminal affinity tag, for specific, rapid purification. The amino terminus was engineered so that, after purification, the unwanted N-terminal tag can be completely removed from the protein by thrombin cleavage, yielding a natural amino acid sequence for each protein. I have cloned the genes for EF-G and all three release factors into this new expression vector and the genes for all the other protein factors into a pCAL-n expression vector. These constructs will allow our laboratory group to quickly and inexpensively purify all the protein factors needed for the new in vitro termination assay. (Abstract shortened by UMI.) ^

THE TRANSLATION PRODUCTS OF MOLONEY MURINE SARCOMA VIRUS 124 GENOMIC RNA

Murine sarcoma viruses constitute a class of replication-defective retroviruses. Cellular transformation may be induced by these viruses in vitro; whereas, fibrosarcomas may result in animals infected with them in vivo (Tooze, 1973; Bishop, 1978). Hybridization studies suggest that murine sarcoma viruses arose by recombination between nondefective murine leukemia virus sequences and certain cellular sequences present in uninfected mouse cells (Hu et al., 1977). A specific gene product, however, has not been implicated in murine sarcoma virus transformation.^ One line of murine sarcoma virus-producing cells, Mo-MuSV-clone 124, (Ball et al., 1973), was studied biochemically because it mainly produces the sarcoma virus as a pseudotype packaged with helper murine leukemia virus proteins. The sarcoma viral RNA was translated in a sophisticated cell-free protein synthesizing system (Murphy and Arlinghaus, 1978). The translation products were analyzed by a number of techniques, including electrophoresis in denaturing gels of SDS polyacrylamide, immunoprecipitation, and peptide mapping. The major products of the total RNA purified from the virus preparation were shown to have molecular weights of about 63,000 (P63('gag)), 42,000 (P42), 40,000 (P40), 38,000 (P38), and 23,000 (P23). The size class of mRNA coding for each of the cell-free products was estimated using a poly(A) selection technique and sucrose gradient fractionation. These analyses were used to localize the coding information related to each of the in vitro synthesized cell-free products within the sarcoma virus genome.^ The major findings of these studies were: (1) the 5' half of the sarcoma viral RNA codes for the 63,000 dalton polypeptide and 42,000 - 38,000 dalton polypeptides derived from the "gag" gene; and (2) the 3' half of the sarcoma viral RNA codes for a 38,000 dalton polypeptide and possibly derived from the cellular acquired sequences. ^

EXPRESSION OF THE HUMAN PLACENTAL LACTOGEN GENES

The placenta is the site of synthesis of various peptide and steroid hormones related to pregnancy. Human placental lactogen (hPL) is the predominant peptide hormone secreted by term placenta and its synthesis is tissue-specific and coupled to placenta development. The objective of this work was to study the structure and expression of the hPL.^ Poly(A('+))RNA from human term placenta was translated in a mouse-derived cell-free system. A major band corresponding to pre-hPL and a minor band comigrating with mature hPL, represent (TURN)15% of the total radioactively labeled proteins. Analysis of the poly(A('+))RNA showed a prominent band at approximately 860 nucleotides. A corresponding band was observed in Northern blots of total RNA, hybridized with {('32)P}-labeled recombinant plasmid containing a portion of hPL cDNA. Similar analyses of nuclear RNA showed at least four additional bands at 990, 1200, 1460 and 1760 nucleotides, respectively, which are likely precursors of hPL mRNA. Poly(A('+))RNA was used to construct a cDNA library, of which approximately 5% of the clones were found to hybridize to hPL DNA sequences. Heteroduplexes constructed between a clone containing a 815 bp hPL cDNA insert and a hPL genomic DNA clone revealed four small intervening sequences which can account for the lengths observed in hnRNA molecules.^ Recombinant plasmid HCS-pBR322 containing a 550 bp insert of a cDNA transcript of human placental lactogen (hPL) mRNA was ('3)H-labeled an hybridized in situ to human chromosome preparations. These experiments allowed assignment of the hPL and growth hormone (hGH) genes, which have over 90% nucleotide homology in their coding sequences, to band q22-24 of chromosome 17. A gene copy number experiment showed that both genes are present in (TURN)3 copies per haploid genome.^ Experiments were designed to determine if all members of the hPL gene cluster, consisting of four non-allelic genes, are transcribed in term placenta. Advantage was taken of differences in restriction endonuclease sites in the coding portions of the different hPL genes, to distinguish the putative cDNAs of the transcriptionally active genes. Two genes were found to be represented in the cDNA library and their cDNA transcripts were isolated and characterized. Three independent methods showed that their corresponding mRNAs are about equally represented in the hPL mRNA population. The two cDNAs code for prehPL proteins which differ at a single amino acid position. However the secreted hPLs have identical amino acid sequences. A tetramer insertion duplication was found in a palindrome area of the 3' untranslated region of one of the hPL mRNAs. ^