The overall objective of this study has been to address some of the longstanding questions concerning functional organization of the interphase nucleus. This was achieved by using recently developed high-resolution fluorescence in situ hybridization techniques for a precise localization of specific DNA and RNA sequences in conjunction with immunocytochemistry and biochemical fractionation. This study is based on the philosophy that new insights may be gained by an approach that attempts to interrelate genomic organization, spatial arrangement of RNA metabolism, and nuclear substructure within the mammalian cell nucleus. The nuclear distribution of an exogenous, viral RNA (Epstein-Barr Virus, EBV) within nuclear matrix preparations was studied by developing an approach which couples in situhybridization with biochemical fractionation procedures. EBV RNA molecules accumulate in highly localized foci or elongated tracks within the nucleus of lymphoma cells. These RNA tracks were retained with spatial and quantitative fidelity in nuclear matrix preparations even after biochemical fractionation which removes 95% of cellular protein, DNA, and phospholipid. This provided direct evidence that the primary transcripts are localized via their binding to, or comprising part of, a non-chromatin nuclear substructure. Then the nuclear distribution of RNA from an endogenous gene, fibronectin, was investigated using fluorescence techniques modified for more sensitive detection of endogenous RNAs within nuclear morphology. A series of in situhybridization experiments were performed using different combinations of intron, cDNA, and genomic probes for RNA/RNA or RNA/DNA analysis in intact cells. Fibronectin RNAs were highly localized in the nucleus, forming foci or tracks. Both intron and exon sequences were highly concentrated at the same site within the nucleus, indicating the presence of primary unspliced transcripts. Double-color hybridization using a nontranscribed 5' flanking sequence probe and a genomic DNA probe showed that the gene and RNA track for fibronectin were spatially overlapped, with the gene consistently towards one end of the track. These results provided evidence that the accumulation of RNA molecules occurs directly at or near the site of transcription, and further indicated a structural polarity to the RNA track formation with the gene towards one end. It was further discovered that within a single cell, cDNA probes produced longer tracks than those formed with intron probes, i.e. intron signals were generally confined to a smaller part of the track than the exon signals, indicating that splicing occurs within the RNA track. Additional experiments using poly(A) RNA hybridization or anti-SC-35 antibody staining combined with fibronectin RNA hybridization have shown that the fibronectin tracks were associated with recently discovered transcript domains enriched in poly(A) RNA and splicing factors. To further determine whether other specific genes and RNAs are functionally organized within the nucleus, the nuclear distribution of several active or inactive genes was analyzed in terms of their spatial relationship to transcript domains. The results indicated that in addition to fibronectin, the genes or their primary transcripts from two other active genes, collagen and actin, were also closely associated with the domains. For both of these, over 90% of the gene/RNA sites were either overlapping or directly contacting the domains. In contrast. for two inactive genes, cardiac myosin heavy chain and neurotensin, it was found that both genes were separated from the domains in the majority of nuclei. Histone genes, which have several unique features, showed a relatively complex result with about half of the gene signals extremely close to the domains. Therefore, three actively expressed genes were demonstrated to be tightly associated with the domains and, moreover, their RNAs showed distinct and characteristic spatial relationships with the domains. In contrast, two inactive genes were not associated with the domains. One potential implication of these finding is that active genes may be preferentially localized in and around these transcript domains. The nuclear localization of another RNA, XIST, standing for X-inactivation specific transcript, was studied because of its potentially unique biological role. XIST is the only gene which is known to be expressed from the inactive human X chromosome but not from the active X chromosome, and was believed to be important in X inactivation. Using fluorescence in situhybridization, it was found that XIST RNA was highly localized within the nucleus and always completely overlapped the Barr body which is the condensed, inactive X chromosome. The different fine distribution pattern of XIST RNA within the nucleus as compared to other protein coding RNAs suggested a unique function for this RNA, possibly involving a structural role in inactivating the X chromosome. The final area of my thesis research was to study and acquire expertise in the applications of fluorescence in situ hybridization in gene mapping and cancer genetics. A retinoblastoma (RB)-related putative tumor suppressor gene, p107, was mapped to human chromosome 20 in band q11.2. Localization of p107 to 20q11.2 was of particular interest because of the correlation of breakpoints in this area with specific myeloid disorders such as acute nonlymphocytic leukemia and myelodysplastic syndrome. Other applications of in situ hybridization including the search for unknown genes at a known chromosomal breakpoint, detection of deletions, translocations or other chromosomal rearrangements associated with specific tumors were also explored and reviewed.

In previous work I described the purification and properties of the microtubule-based mechanochemical ATPase cytoplasmic dynein. Cytoplasmic dynein was found to produce force along microtubules in the direction corresponding to retrograde axonal transport. Cytoplasmic dynein has been identified in a variety of eukaryotes including yeast and human, and there is a growing body of evidence suggesting that this "molecular motor" is responsible for the transport of membranous organelles and mitotic chromosomes. The first part of this thesis investigates the molecular basis of microtubule-activation of the cytoplasmic dynein ATPase. By analogy with other mechanoenzymes, this appears to accelerate the rate-limiting step of the cross-bridge cycle, ADP release. Using limited proteolysis, site-directed antibodies, and N-terminal microsequencing, I identified the acidic C-termini of α and β-tubulin as the domains responsible for activation of the dynein ATPase. The second part of this thesis investigates the structure of the 74 kDa subunit of cytoplasmic dynein. The amino acid sequence deduced from cDNA clones predicts a 72,753 dalton polypeptide which includes the amino acid sequences of nine peptides determined by microsequencing. Northern analysis of rat brain poly(A) revealed an abundant 2.9 kb mRNA. However, PCR performed on first strand cDNA, together with the sequence of a partially matching tryptic peptide, indicate the existence of three isoforms. The C-terminal half is 26.4% identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70 kDa subunit of flagellar outer arm dynein. Based on what is known about the Chlamydomonas70 kDa subunit, I suggest that the 74 kDa subunit is responsible for targeting cytoplasmic dynein to membranous organelles and kinetochores of mitotic chromosomes. The third part of this thesis investigates a 50 kDa polypeptide which co-purifies with cytoplasmic dynein on sucrose density gradients. Monoclonal antibodies were produced against the 50 kDa subunit and used to show that it is a component of a distinct 20S complex which contains additional subunits of 45 and 150 kDa. Moreover, like cytoplasmic dynein, the 50 kDa polypeptide localizes to kinetochores of metaphase chromosomes by light and electron microscopy. The 50 kDa-associated complex is reported to stimulate cytoplasmic dynein-mediated organelle motility in vitro. The complex is, therefore, a candidate for modulating cytoplasmic dynein activity during mitosis.

DNA is packaged within the cells' nucleus as a highly compact chromatin structure ranging between 100-400 nm fibers. The organization and alteration of this structure is mandatory in order to arbitrate DNA-mediated processes of the cell, including transcription, DNA replication, recombination and repair. Many different kinds of enzymes modify chromatin components and, in turn, regulate the accessibility of DNA. These multi-subunited enzymes have emerged as key regulators for several processes of the cell. Central to understanding how DNA-mediated processes are regulated is to comprehend the consequences of these modifications of chromatin, which lead to altered states of either activation or inactivation. One class of factors known to modify chromatin structure is the ATP-dependent chromatin remodeling enzymes. This class of enzymes encompasses evolutionarily conserved multi-subunited enzymes, which appear to function by using the energy of ATP hydrolysis to disrupt histone-DNA interactions. The prototype of ATP-dependent chromatin remodelers is the Saccharomyces cerevisiae SWI/SNF complex. The yeast SWI/SNF complex is required for the full functioning of several transcriptional activators and for the expression of a subset of yeast genes, a notable number being inducible and mitotic genes. The purified complex is comprised of the following eleven different polypeptides: Swi2p/Snf2p, Swi1p, Swi3p, Snf5p, Snf6p, Swp73p, Arp7p, Arp9p, Swp82p, Swp29p and Snf11 p. It has been established that a core of homologous subunits (Swi2p, Swi3p, Swp73p, Snf5p and the Arp proteins) is conserved among the SWI/SNF-related complexes from several organisms (yRSC, hSWI/SNF, hRSC, DrosophilaBrahma). However, the functional contribution of these polypeptides in the complexes for altering chromatin structure is largely unknown. In this study, biochemistry is used to examine the structure of the complex and function of individual subunits of the yeast SWI/SNF complex to understand better how these proteins are acting in concert to remodel chromatin. In addition, we examine a role for SWI/SNF complex in the process of DNA replication. The relative stoichiometry of the SWI/SNF complex subunits was determined by in vitrobiochemical studies. Co-immunoprecipitation has demonstrated that there is only one copy of Swi2p/Snf2p per complex. Subsequent radioactive labeling of the purified complex revealed that the complex contains one copy of each subunit per complex with the exception of Swi3p and Snf5p, which are present in two copies per complex. The subunit organization of SWI/SNF complex has been more clearly defined by determining direct subunit-subunit interactions in the complex. The Swi3p component has previously been shown to be critical for complex function in vivo and essential for the integrity of the complex in vitro, and this study demonstrates that Swi3p serves as a scaffolding protein that nucleates SWI/SNF complex assembly. In vitrobinding studies with Swi3p have revealed that Swi3p displays self-association, as well as direct interactions with the Swi2p, Snf5p, Swp73p, Swi1p and Snf6p members of the complex. The direct interactions of the yeast SWI/SNF subunits with transcriptional activators, thought to be important for yeast SWI/SNF targeting, were examined. In vitrobinding assays demonstrate that individual SWI/SNF subunits, Snf5p, Snf6p and Swi1p, and sub-complexes Swi2p/Swi3p and Swp73p/Swi3p can directly interact with specific domains of transcriptional activators of either the Swi5p zinc-finger DBD or VP16 acidic activation domain. This work begins to characterize the functional contribution of individual subunits, and cooperative sub-complexes that are critical for the SWI/SNF complex functional activities. The yeast SWI/SNF complex was investigated for the ability to playa role in DNA replication. Interestingly, plasmid stability assays reveal that minichromosomes that contain DNA replication origin ARS121 is weakened when the SWIISNF complex is non-functional. ARS121's SWI/SNF dependency is overcome by the over-expression of DNA replication regulatory protein, Cdc6p. Thus, this suggests SWI/SNF may either indirectly effect DNA replication by effecting the expression of Cdc6p, or has a redundant function with Cdc6p. In addition, several crippled derivatives of ARS1 acquire SWI/SNF dependence, and it is found that the SWI/SNF complex requires a transcriptional activation domain to enhance ARS1 function. These results reinforce the view that SWI/SNF play a role in two chromatin-mediated processes', transcription and DNA replication.

Gene transfer plays an important role in bacterial evolution. Especially in an under explored species like Rhodococcus, a type of bacteria found in the soil. Rhodococcus has several applications in the pharmaceutical industry and in the production of antibiotics. Rhodococcus possess several unique sets of properties which makes it beneficial to have a reliable method of producing mutants of Rhodococcus. The goal of the experiment was to find an efficient way of forming Rhodococcus colonies with kanamycin resistant genes. The project began from an unexpected observation from an earlier experiment with Rhodococcus strain MTM3W5.2. where I attempted to transform this strain with a transposon via electro-transformation. The colonies that grew/ appeared transformants were screened to confirm the presence of kanamycin gene, however there was no amplified DNA seen on the PCR gel (i.e. absence of the kanamycin gene). The electro-transformant colonies were selected on LB plates containing different higher concentrations of kanamycin. Then the appeared transformants were again screened via disk diffusion assay and were classified into 3 different kanamycin resistant phenotypes. Majority of the “C” phenotypic colonies (i.e., high level resistance to kanamycin) appear to contain the kanamycin gene, but these colonies were less in numbers. This led us to try another method of gene transfer which is conjugation. Conjugation was carried on a double selection antibiotic plate containing both chloramphenicol (30 µg) and kanamycin (100 µg). The transconjugate colonies that appeared on the double selection plates were also screened by PCR, but none of the colonies had amplified DNA suggesting absence of the kanamycin gene. The colonies seen on the double selection plate were possibly due to spontaneous mutation or some type of unknown phenotypic variation. However, in the future, double selection plates with higher concentrations of antibiotics can possibly give us transconjugants with kanamycin genes.

No description available.

Common, complex disorders are polygenic and multifactorial traits representing interactions between environmental, genetic and epigenetic risk factors. More often than not, contributions of these risk factors have been studied individually and this is especially true for complex reproductive traits where application of genomic technologies has been challenging and slow to progress. This thesis explores the potential of genetic and epigenetic components contributing to a better understanding of the biological pathways underlying disease risk in two specific female complex reproductive traits - polycystic ovary syndrome (PCOS) and preterm premature rupture of membranes (PPROM). The PCOS projects focus on characterization of a gene, DENND1A, whose association to PCOS has been established by Genome Wide Association Studies (GWAS) and is known to contribute to PCOS steroidogenic phenotype. In addition, differential microRNAs expression contributing to DENND1A expression regulation in PCOS theca cells was identified. The studies on PPROM utilize a Whole Exome Sequencing approach to identify rare variants in fetal genes contributing to extracellular matrix composition and synthesis contributing to PPROM risk. The results suggest that fetal contribution to PPROM is polygenic and is driven by a significant genetic burden of potentially damaging rare variants in genes contributing to fetal membrane strength and integrity. Tissue and location specific expression patterns of the Chromosome 21 miRNA cluster (miR-99a, miR-125b, let-7c) in fetal membranes from term pregnancies with spontaneous rupture were investigated. The results suggest that these miRNAs play potential roles in fetal membrane rupture and fetal membrane defects associated with T21.

In the inner and outer hair cells (OHCs) of the inner ear, an unconventional myosin 15a localizes at the tips of mechanosensory stereocilia and plays an important role in forming and maintaining their normal structure. A missense mutation makes the motor domain of myosin 15a dysfunctional and is responsible for the congenital deafness DFNB3 in humans and deafness and vestibular defects in Shaker-2 (Sh2) mouse model. All hair cells of homozygous Shaker-2 mice (Myo15sh2/sh2) have abnormally short stereocilia, but, only stereocilia of Myo15sh2/sh2OHCs start to degenerate after the first few days of postnatal development and lose filamentous tip links between stereocilia that are crucial for mechanotransduction. The exact mechanisms of this degeneration are unknown even though they may underlie DFNB3 deafness in humans. We hypothesize that structural abnormalities in Myo15sh2/sh2 OHCs may alter the mechanical forces applied to the mechano-electrical transduction (MET) channels resulting in abnormal ionic homeostasis, which may lead to eventual degeneration of Myo15sh2/sh2 OHCs. Therefore, we investigated the ionic conductances and integrity of mechanotransduction apparatus in Myo15sh2/sh2 OHCs. Surprisingly, we found that myosin 15a-deficiency is associated not only with structural abnormalities of OHC stereocilia but also with alterations of voltage-gated ion conductances.

Neonatal mice have immature immune systems with defects in several components of inflammatory, innate, and specific immune responses and develop a preferential T helper type 2 (Th2) response following immunization with many vaccine antigens. Although maternal antibody is the major form of protection from disease in early life when the neonatal immune system is still immature, the presence of maternal antibody also interferes with active immunization, placing infants at risk for severe bacterial and viral infection. Recent studies have suggested that immunizing with DNA plasmids encoding the vaccine antigen of interest is highly efficacious in a variety of adult animal models. However, similar extensive studies have not been conducted in infants. In this dissertation, we examine both the quantitative and qualitative differences between neonatal and adult humoral and cell-mediated immune responses in the presence or absence of maternal antibody. First, we wished to determine if one-day-old neonatal mice immunized with plasmid DNA expressing influenza A/PR/8/34 hemagglutinin (HA) by either intramuscular (i.m.) or gene gun (g.g.) inoculation were capable of generating humoral responses comparable to those in mice immunized as adults. We found that newborn mice developed stable, long-lived, protective anti-HA-specific IgG responses similar in titer to those of adult DNA-immunized mice. However, unlike the adult i.m. and g.g. DNA immunizations, which develop polarized IgG2a and IgG1 responses, respectively, mice immunized as neonates developed a variety of IgG1-, IgG2a-, and mixed IgG1/IgG2a responses regardless of the inoculation method. Boosting increased, but did not change these antibody profiles. We also found that, in contrast to the DNA immunizations, inoculations of newborn mice with an A/PR/8/34 viral protein subunit preparation failed to elicit an antibody response. Further, temporal studies revealed that both responsiveness to protein vaccination and development of polarized patterns of T help following DNA immunization appeared by 2 weeks of age. To determine if the disparity of polarized IgG responses between neonatal and adult DNA vaccinated mice was due to deficiencies in Th1 promoting cytokines, we addressed the ability of DNA encoding Th1 cytokines to bias the isotype of antibody raised by neonatal DNA immunization. We found that neonatal mice coimmunized with HA and either IL-12 or IFNγ-expressing DNAs developed IgG2a-biased immune responses, regardless of inoculation method, whereas these DNAs had no effect on IgG subtype patterns in adult DNA immunized mice. Consistent with the Th1-promoting effects of these cytokines, we also observed that codelivery of IL-12 or IFNγ DNAs raised T helper responses toward Th1 in mice immunized both as neonates or adults. Thus, codelivery of cytokine DNAs may be effective at tailoring immune responses depending on the required correlates of protection for a given pathogen. Finally, we addressed the effect of maternal antibody on the elicitation of humoral and cell-mediated immune responses. We tested the ability of i.m. and g.g. immunization with DNA expressing influenza HA and/or nucleoprotein (NP) to raise protective humoral and cellular responses in the presence and absence of maternal antibody. We found that neonatal mice born to influenza-immune mothers raised full antibody responses to NP but failed to generate antibody responses to HA. In contrast, the presence of maternal antibody did not affect the generation of long-lived CD4+ and CD8+ T cell responses to both HA and NP. Thus, maternal antibody did not affect cell-mediated responses, but rather it limited humoral responses, with the ability to limit the antibody response correlating with whether the DNA-expressed immunogen was localized in the plasma membrane or within the cell. We further observed that protection from influenza virus challenge was dependent on the presence of anti-HA IgG and was independent of the presence T cell responses. Taken together with other published studies, the data presented in this dissertation help better characterize the responses elicited by DNA vaccines at birth. This dissertation presents several novel observations including the temporal development of polarized IgG subtype responses, the ability of codelivered Th1 cytokine DNA to affect both antibody and T cell responses in the neonate, and the ability to generate humoral responses to intracellular, but not plasma membrane proteins, in the presence of maternal antibody. Furthermore, the data provides rationale for further development of DNA vaccines in the neonate.

Dynactin is a multi-subunit complex which was initially identified in 1991 as an activator of cytoplasmic dynein-driven microtubule-based organelle motility in vitro. Although genetic studies also supported the involvement of both complexes in the same functional pathways in yeast, filamentous fungi, and Drosophila, none of these findings yielded significant insights into dynactin's mechanism of action. The full range of cytoplasmic dynein functions in vertebrate cells has also remained poorly understood, due, in large part, to the lack of a specific method of inhibition. The present thesis work was designed to investigate these issues through a study of the 50 kDa subunit of dynactin. As a first step (Chapter 1), I cloned mammalian p50 and characterized its expression at the tissue and subcellular levels. Rat and human cDNA clones revealed p50 to be a novel α-helix-rich protein containing several highly-conserved structural features including one predicted coiled-coil domain. Immunofluorescence staining of p50, as well as other dynactin and cytoplasmic dynein components in cultured vertebrate cells showed that both complexes are recruited to kinetochores during prometaphase and concentrate near spindle poles thereafter. These findings represented the first evidence for dynactin and cytoplasmic dynein co-localization within cells, and for the presence of dynactin at kinetochores. The second major phase of the thesis (Chapter 2) was focused on investigating dynactin and cytoplasmic dynein function in cultured cells in vivo using a dominant negative inhibition approach based on transient transfections of p50 constructs. Overexpression of wild type human p50 in cultured cells resulted in a dramatic fragmentation and dispersal of the Golgi apparatus. Time-lapse fluorescence microscopy analysis of p50-overexpressing cells revealed that microtubule-based vesicle transport from the endoplasmic reticulum to the Golgi was inhibited. Also, the interphase microtubule organizing center was found to be less well-focused in some but not all transfected cells. Overexpression of p50 also disrupted mitosis, causing cells to accumulate in a prometaphase-like state. Chromosomes were condensed but unaligned, and spindles, while still generally bipolar, were dramatically distorted. Sedimentation analysis revealed the dynactin complex to be dissociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores was markedly diminished in cells expressing high levels of p50. These findings provided the first in vivoevidence for the role of dynactin in cytoplasmic dynein function, i.e. mediating the motor's binding to at least one "cargo" organelle, the kinetochore, and probably also to others such as vesicles destined for the Golgi complex. These data also strongly implicated both dynactin and dynein in Golgi organization during interphase, and chromosome alignment and spindle organization during mitosis. Based on the remarkable disruptive phenotypic effects associated with overexpressing of p50, the name of dynamitin was proposed for this polypeptide. In the third and last phase of the thesis (Chapter 3), two issues were addressed: first, the dynamitin-induced mitotic arrest phenotype was studied in greater detail to better understand the exact sites of dynactin and cytoplasmic dynein activity throughout mitosis. Second, a domain analysis of dynamitin was performed to gain insight into its function within the dynactin complex. A time-lapse fluorescence microscopy study of mitosis in living dynamitin-overexpressing COS-7 cells strongly suggested specific defects in interactions of astral microtubules with the cell cortex, and in both spindle pole assembly and maintenance. Analysis of the mitotic arrest phenotype in a second cell line revealed a second arrest point at metaphase, and a clear effect of dynamitin overexpression on spindle axis orientation, again consistent with defects in interactions between microtubules and the cell cortex. Refined analyses of kinetochore and spindle pole components also confirmed specific defects in kinetochore function and spindle pole organization. Taken together, these findings support three main sites of dynactin and cytoplasmic dynein activity during vertebrate mitosis: prometaphase kinetochores, spindle poles, and the cell cortex. Finally, the domain analysis revealed dynamitin to be capable of self-association through at least two separate interaction domains, consistent with models of the mechanism underlying dynamitin-induced dynactin dissociation, and therefore, yielding important new insights into dynactin assembly. This study also indicated that a third region within dynamitin, residues 105 to 154, is essential for dynamitin and dynactin function. An independent study confirmed this finding, implicating this region in binding to ZW10, an upstream kinetochore protein. Dynamitin has therefore been revealed to be the kinetochore-targeting subunit of dynactin, and indirectly, cytoplasmic dynein. Through the body of this thesis work, dynamitin has also emerged as a powerful new tool for studying vertebrate dynactin and cytoplasmic dynein function in vivo and in vitro.

RNA interference (RNAi) in the nematode Caenorhabditis elegans is a type of homology-dependent post-transcriptional gene silencing induced by dsRNA. This dissertation describes the genetic analysis of the RNA interference pathway and inheritance properties associated with this phenomenon. We demonstrate that the RNAi effect can be observed in the progeny of the injected animal for at least two generations. Transmission of the interference effect occurs through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-l and rde-4 are required for the formation of this interfering agent but are not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes are required downstream for interference. These findings provide evidence for germline transmission of an extragenic sequence-specific silencing factor and implicate rde-l and rde-4in the formation of the inherited agent. Other forms of homology-dependent silencing in C. elegansinclude co-suppression and transcriptional silencing of transgenes in the germline. We demonstrate that silencing of a germline transgene can be initiated by injected dsRNA, via the RNAi pathway, and then maintained on a different level. This observation indicates that post-transcriptional and transcriptional silencing of homologous genes could be connected. This dissertation also describes the connection between RNAi and developmental pathways of gene regulation in C. elegans. We show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-l), and two homologs of rde-1 (alg-l and alg-2) cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-l, alg-l, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7small temporal RNAs that regulate stage-specific development. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation. Finally, this study illustrates the detection of small interfering RNAs (siRNAs), intermediates in the RNAi process, and describes requirements for their accumulation. We show that, in the course of RNAi induced by feeding dsRNA, C. elegans accumulate only siRNAs complementary to the target gene. This accumulation depends on the presence of the target sequence and requires activities of several RNAi-pathway genes. We show that selective retention or amplification of RNAi-active molecules can create a reservoir of memory antisense siRNAs that prevent future expression of the genes with complementary sequence. This suggests a parallel at the molecular level with the clonal selection of antibody forming cells and in the vertebrate immune system.