The Caenorhabditis elegans gene gro-1 belongs to the Clock group of genes. The four known genes making up this class are believed to be involved in a general mechanism acting to coordinate the time-dependent processes in the organism. Mutation of these genes alter the timing of many disparate processes. This results in the mean lengthening of embryonic and post-embryonic development, as well as in a lengthening of the periods of a number of adult behaviours including pharyngeal pumping, swimming and the defecation cycle. These mutants also exhibit a significantly increased life span. The gene gro-1 has been cloned and encodes a metazoan N6-(Delta2) isopentenyl PPi: tRNA isopentenyl transferase. In S. cerevisiae and bacteria this enzyme has been shown to modify tRNAs that code for codons begining with U. This modification consists of the isopentenylation of the adenine residue at position 37, adjacent to the anti-codon. Interestingly, gro-1 is the fifth member of an operon. Preliminary expression studies with GFP reporter constructs suggest that as in yeast, GRO-1 is expressed in the cytoplasm and mitochondria, as well as in the nucleus.

The gene clk-2 of C. elegans is required in both the germline and the soma, for subsequent embryonic viability, and for developmental and behavioural rates, respectively, clk-2 encodes a protein that is homologous to Tel2p in yeast, which is required for the telomere length regulation. It has been demonstrated that clk-2 affects telomere length also in worms and human cells. By now the exact biochemical function of CLK-2 is unknown. In order to shed light onto the function of the gene clk-2, a two-hybrid screen was carried out to identify the interactors of the protein CLK-2. A potential interactor of CLK-2, Y105C5B.19, was identified in the screen. Y105C5B.19 is a novel gene and does not have homologues in other species. Y105C5B.19 contains an MSP (major sperm protein) domain, therefore it is possible that it could be involved in the processes that regulate oocyte maturation, gonadal sheath contractions, or sperm mobility. Interestingly, given that clk-2 is required for subsequent embryogenesis at some point during a narrow time between the end of oocyte maturation and the 2-cell stage, it is tempting to speculate that the interaction between CLK-2 and Y105C5B.19 might be functionally relevant. The lethality of clk-2(qm37) mutants might result from delayed consequences of defects in ovulation and/or fertilization, and perhaps such defects could result from the disruption of the interaction between CLK-2 and Y105C5B.19. The amino acid substitution C772Y resulting from the clk-2(qm37) mutation was found to disrupt the interaction between CLK-2 and Y105C5B.19 in a two-hybrid assay, lending support to the idea that the interaction between CLK-2 and Y105C5B.19 takes place in vivo.

In Caenorhabditis elegans, mutations in the clk-1 gene result in delayed embryonic and post-embryonic development, a slowing down of rhythmic behaviors and an extended life span. CLK-1 encodes the demethoxyubiquinone (DMQ or DMQn) hydroxylase in the ubiquinone (CoQ or Qn) biosynthesis pathway. Thus, clk-1 mutants produce DMQ instead of CoQ. In order to understand the relationship between the CoQ biosynthesis defect and the pleiotropic phenotype of clk-1 mutants, I isolated a deletion mutant, coq-4(qm143), in C. elegans. In Saccharomyces cerevisiae, mutants in COQ4, the coq-4 homologue, do not produce ubiquinone, like those in COQ7, the clk-1 homologue. coq-4(qm143) is a non-strict maternal-effect lethal mutation. Most of the progeny from a homozygous coq-4(qm143) hermaphrodite die during embryogenesis. However, homozygous coq-4(qm143) hermaphrodites from a heterozygous mother can develop and behave normally until adulthood. As adults, they become uncoordinated and paralytic, and are defective in egg-laying. Unlike hermaphrodites, homozygous coq-4(qm143) males are fully maternally rescued. The qm143 is a 1469 base pair deletion, which completely removes the coq-4 gene and does not affect the coding sequence of any other gene. By performing germline transformation, I also showed that the non-viable phenotype of coq-4 (qm143) is indeed due to the removal of the coq-4 gene itself. The preliminary study of COQ-4 expression pattern by using a COQ-4::GFP fusion protein indicates that COQ-4 is expressed in mitochondria of the worm.

In an effort to find new genes involved in development and the biological timing, I carried out a new screen for viable maternal-effect mutations similar in protocol to the previous screen in which 24 such genes have been identified. I screened 10,600 genomes and isolated 6 slow-growing mutations and 5 behavioral and morphological mutations. Another maternal-effect slow-growing mutation is isolated from a screen for both maternal-effect and non maternal-effect slow development mutations. Genetic mapping suggests that none of the seven slow growing mutations corresponds to previously identified genes, so seven new clk genes (clk-4, clk-5, clk-6, clk-7 clk-8, clk-9, clk-10) have been identified. Because most identified clk genes (clk-2, clk-4 to clk-10 and gro-1) are defined by single allele, we believe that these genes have not yet been saturated. Mutants of seven new clk genes have typical Clk phenotypes: a mean lengthening of embryonic development, postembryonic development, defecation cycle and life span. As the screening procedure did not involve any measure of life span, it is suggested that slow life is sufficient for long life. As expected, all seven mutations can be maternally rescued.

The nematode worm Caenorhabditis elegans has become a model system for the analysis of the genetics of aging. Previously, mutations in four genes, age-1, clk-1, daf-2 and daf-28 had been shown to lengthen adult life span. Based on the molecular genetic analysis of these genes, the sole function of the dauer genes age-1, daf-2 and possibly daf-28 is to regulate the activity of the forkhead-like transcription factor daf-16. daf-16 may determine life span by regulating the transcription of genes that are necessary for resistance to stresses, especially oxidative stress. Mutations in clk-1 affect behavioral and developmental timing as well as increasing mean and maximum life span. I show that mutations in the genes clk-2, clk-3 and gro-1 affect many of the same processes as clk-1 and that these four genes interact to determine the length of development and adult life span. These four Clock genes lengthen life span in a manner that is distinct from that of the dauer genes. clk-1 has been cloned and has been implicated in the regulation of metabolism. This suggests that Clock mutants may live long because they have reduced metabolic rates. I also show that mutations in 7 genes that affect feeding behavior, eat-1, eat-2, eat-3, eat-6, eat-13, eat-18 and unc-26 lengthen life span. This effect is presumably due to reduced caloric intake (caloric restriction) which has been shown to lengthen the life span of a wide variety of animals. eat-2 lengthens life span by a mechanism that is distinct from that of the dauer mutants but may be similar to that of the Clock mutants. This suggests that caloric restriction may also reduce metabolic rates, possibly through down-regulation of the Clock genes. These results indicate that life span in C. elegans is a polygenic trait, influenced by many different physiological processes. The study of genes that affect aging in C. elegans provides support for the antagonistic pleiotropy and free radical theories of aging.

Rapid progress has been made toward understanding the significance of CDK inhibitor proteins (CKIs) in the regulation of cell cycle progression. The overall goal of this study has been targeted to further expand our knowledge of CKI function through the investigation of a previously uncharacterized CKI named cki-2 during development in C. elegans. The characterization of cki-2 using a reverse genetic approach called co-suppression has revealed a novel mechanism that cki-2 and its related cell cycle regulators are required for the appropriate elimination of centrioles during oogenesis. Loss of cki-2 in the germ line caused perdurance of centrioles into the one-cell embryo, resulting in supernumerary centrosomes and aberrant cell divisions in the first cell cycle. This was significantly suppressed by reduction of cyclin E and a Cdk2 homologue, indicating that these cell cycle regulators are involved in this critical developmental process. In order to further understand the function of cki-2, a yeast two-hybrid screen was conducted which allowed us to identify three CKI-2 interacting proteins: orthologues of PCNA (PCN-1), SUMO (SMO-1), and a RING finger protein called RNF-1. CKI-2 has functionally separable domains in its amino (Cyclin/Cdk binding)- and carboxy (PCNA binding)-terminus and they exert distinct roles in cell cycle progression. It was observed that CKI-2 is covalently modified by SUMO on its N-terminus and this causes CKI-2 to relocalize to thr nucleolus, which is associated with its rapid degradation. Since many RING finger proteins act as components of the multi-subunit E3 ubquitin ligases, we speculated that RNF-1 might be involved in the CKI-2 degradation. This possibility was tested by co-expression of RNF-1 with CKI-2, revealing that co-expression of RNF-1 suppresses the embryonic lethality caused by the CKI-2 overexpression and moreover, this is correlated with an increased rate of CKI-2 degradation. In addition, western blot analyses performed on different genetic backgrounds suggested that the CKI-2 degradation occurs in an ubiquitin-dependent manner through the proteasome-mediated proteolysis pathway. Furthermore, a yeast-based assay developed to test a possible role of SUMO in modulating the CKI-2/RNF-1 interaction demonstrated that SUMO may antagonize the interaction between CKI-2 and RNF-l, these highlighting an intriguing model that appropriate levels of CKI-2 are regulated through ubiquitin-dependent proteolysis mediated by RNF-l, and which maybe modulated by SUMO.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 122-131). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 122-131). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (p. [110]-117).

Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, Dec. 2005. / Title from title screen. Includes bibliographical references.