Role of ovarian cancer antigen CA125/MUC16 in development and cancer

Cancer antigen 125 (CA125) is a tumor antigen that is routinely used to monitor the disease progress and the outcome of treatment in ovarian cancer patients. Elevated serum levels of CA125 are detected in over 80% of epithelial ovarian cancer patients. CA125 is a high molecular weight (>1M Dalton) mucin-type glycoprotein encoded by the MUC16 gene on human chromosome 19. Although MUC16 has served as the best serum marker for monitoring growth of ovarian cancer, roles for MUC16 in normal physiology and ovarian cancer are largely unknown. To understand the biological functions of MUC16, I characterized a mouse Muc16 homolog on chromosome 9 by means of expression pattern profiling, phenotype analysis of Muc16 knockout mice, and in vitro and in vivo studies of Muc16 null transformed ovarian surface epithelial (OSE) cells. ^ The mouse Muc16 homolog shares a conserved genomic structure with human MUC16. In addition to being expressed in mouse ovarian cancer, mouse Muc16 mRNA and protein were expressed in the mesothelia covering the heart, lung, ovary, oviduct, spleen, testis, and uterus. The conserved genomic structure and expression pattern of mouse Muc16 to human MUC16 suggests that mouse Muc16 is the ortholog of human MUC16. To understand the biological functions of Muc16, I generated Muc16 knockout mice. Muc16 knockout mice were viable, fertile and normal by one year of age. However, between 18 and 24 months of age, Muc16 knockout mice developed various tissue abnormalities such as ovarian cysts and tumors of the liver and other peritoneal organs. To determine the role of MUC16 in ovarian cancer progression, I established Muc16 null transformed ovarian surface epithelial (OSE) cell lines, following the same method to develop mouse model of epithelial ovarian cancer (Orsulic et al., 2002). Loss of Muc16 did not affect cell morphology, cell proliferation rate, or tumorigenic potential. However, Muc16-null OSE cells showed decreased attachment to extracellular matrix proteins as well as to primary mouse peritoneal mesothelial cells. Peritoneal mesothelia are the most frequent implantation sites of ovarian cancer. Furthermore, a pilot transplantation assay suggests that Muc16 null transformed OSE cells formed less disseminated tumors in the peritoneal cavity compared to wild-type OSE cells. ^ In conclusion, these results demonstrate that MUC16 is not required for normal mouse development or reproduction, but plays important roles in tissue homeostasis, ovarian cancer cell adhesion and dissemination. This study provides the first in vivo evidence of the roles of MUC16 in development, as well as ovarian cancer progression and dissemination. These studies offer valuable insights into possible mechanisms of ovarian cancer development and potential molecular targets for ovarian cancer treatment. ^

SYSTEMIC TOXICITY IN RODENTS FOLLOWING ACUTE AND SUBCHRONIC INHALATION OF FORMALDEHYDE (FLOW CYTOMETRY, CELL CYCLE KINETICS, ALKALINE ELUTION)

Systemic toxicity was evaluated in Sprague-Dawley (SD) rats and A-strain mice exposed to HCHO inhalation at 0, 0.5, 3, or 15 ppm for six hours/day, five days/week for up to 24 weeks. Toxicity was measured by flow cytometry to detect changes in cell cycle RNA and DNA content and by alkaline elution to detect DNA protein cross-link (DPC) formation.^ A G(,2)M block was detected in SD rat marrow following one week of exposure to 0.5, 3, or 15 ppm HCHO, but this block did not persist. No effect was noticed in mouse marrow. Only a minimal increase in RNA content was detected in rat or mouse marrow while exfoliated lung cells showed a significant increase in RNA activity after one week of exposure.^ Acute exposure in SD rats for four hours/day for one or three days at 150 ppm showed an increase in RNA activity in exfoliated lung cells but not in the marrow after one day. On the third day, dead cells were detected in exfoliated lung cells.^ In alkaline elution studies, no DPC were detected in marrow of SD rats after 24 weeks exposure up to 15 ppm. During acute exposures, a dose response relationship was detected in SD rat exfoliated lung cells which yielded cross-linking factors of 0.954, 1.237, and 1.417 following a four hour exposure to 15, 50, or 150 ppm, respectively. No DPC were detected in the marrow at 150 ppm. In vitro exposures to HCHO of CHO and SHE cells and rat marrow cells revealed the production of DPC and DNA-DNA cross-links.^ Cytoxan treatment of SD rats was used to provide positive controls for flow cytometry and alkaline elution. A drastic reduction in RNA content and cycling cells occurred one day following treatment. After four days, RNA content was greatly increased; and on day eleven the marrow had regenerated. DPCs were detected in both the marrow and the exfoliated lung cells.^ The lack of significant responses in SD rats and A-strain mice below 15 ppm HCHO is explainable by host defense mechanisms. Apparently, the mucociliary apparatus and enzymatic detoxification are sufficient to reduce systemic toxicity to low level concentrations of formaldehyde. ^

GENOME-WIDE PROFILING UNVEILS CRITICIAL FUNCTIONS OF p53 IN HUMAN EMBRYONIC STEM CELLS

Embryonic stem cells (ESCs) possess two unique characteristics: infinite self-renewal and the potential to differentiate into almost every cell type (pluripotency). Recently, global expression analyses of metastatic breast and lung cancers revealed an ESC-like expression program or signature, specifically for cancers that are mutant for p53 function. Surprisingly, although p53 is widely recognized as the guardian of the genome, due to its roles in cell cycle checkpoints, programmed cell death or senescence, relatively little is known about p53 functions in normal cells, especially in ESCs. My hypothesis is that p53 has specific transcription regulatory functions in human ESCs (hESCs) that a) oppose pluripotency and b) protect the stem cell genome in response to DNA damage and stress signaling. In mouse ESCs, these roles are believed to coincide, as p53 promotes differentiation in response to DNA damage, but this is unexplored in hESCs. To determine the biological roles of p53, specifically in hESCs, we mapped genome-wide chromatin interactions of p53 by chromatin immunoprecipitation and massively parallel tag sequencing (ChIP-Seq), and did so under three VIdifferent conditions of hESC status: pluripotency, differentiation-initiated and DNA-damage-induced. ChIP-Seq showed that p53 is enriched at distinct, induction-specific gene loci during each of these different conditions. Microarray gene expression analysis and functional annotation of the distinct p53-target genes revealed that p53 regulates specific genes encoding developmental regulators, which are expressed in differentiation-initiated but not DNA- damaged hESCs. We further discovered that, in response to differentiation signaling, p53 binds regions of chromatin that are repressed but also poised for rapid activation by core pluripotency factors OCT4 and NANOG in pluripotent hESCs. In response to DNA damage, genes associated with migration and motility are targeted by p53; whereas, the prime targets of p53 in control of cell death are conserved for p53 regulation in both differentiation and DNA damage. Our genome-wide profiling and bioinformatics analyses show that p53 occupies a special set of developmental regulatory genes during early differentiation of hESCs and functions in an induction-specific manner. In conclusion, our research unveiled previously unknown functions of p53 in ESC biology, which augments our understanding of one of the most deregulated proteins in human cancers.

Acceleration of the PanIN Development in Mice Expressing Oncogenic K-Ras Due to a High Fat Diet

Obesity is postulated to be one of the major risk factors for pancreatic cancer, and recently it was indicated that an elevated body mass index (BMI correlates strongly with a decrease in patient survival. Despite the evident relationship, the molecular mechanisms involved are unclear. Oncogenic mutation of K-Ras is found early and is universal in pancreatic cancer. Extensive evidence indicates oncogenic K-Ras is not entirely active and it requires a triggering event to surpass the activity of Ras beyond the threshold necessary for a Ras-inflammation feed-forward loop. We hypothesize that high fat intake induces a persistent low level inflammatory response triggering increased K-Ras activity and that Cox-2 is essential for this inflammatory reaction. To determine this, LSL-K-Ras mice were crossed with Ela-CreER (Acinar-specific) or Pdx-1-Cre (Pancreas-specific) to “knock-in” oncogenic K-Ras. Additionally, these animals were crossed with Cox-2 conditional knockout mice to access the importance of Cox-2 in the inflammatory loop present. The mice were fed isocaloric diets containing 60% energy or 10% energy from fat. We found that a high fat diet increased K-Ras activity, PanIN formation, and fibrotic stroma significantly compared to a control diet. Genetic deletion of Cox-2 prevented high fat diet induced fibrosis and PanIN formation in oncogenic K-Ras expressing mice. Additionally, long term consumption of high fat diet, increased the progression of PanIN lesions leading to invasive cancer and decreased overall survival rate. These findings indicate that a high fat diet can stimulate the activation of oncogenic K-Ras and initiate an inflammatory feed forward loop requiring Cox-2 leading to inflammation, fibrosis, and PanINs. This mechanism could explain the relationship between a high fat diet and elevated risk for pancreatic cancer.

Biological effects of PEA3 expression in {\it HER-2\/}/{\it neu\/}-overexpressing human cancer cells and the molecular mechanisms of PEA3-mediated transcriptional repression on {\it HER-2\/}/{\it neu\/}

HER-2/neu is a receptor tyrosine kinase highly homologous with epidermal growth factor receptor. Overexpression and/or amplification of HER-2/neu has been implicated in the genesis of a number of human cancers, especially breast and ovarian cancers. Transcriptional upregulation has been shown to contribute significantly to the overexpression of this gene. Studies on the transcriptional regulation of HER-2/neu gene are important for understanding the mechanism of cell transformation and developing the therapeutic strategies to block HER-2/neu-mediated cancers. PEA3 is a DNA binding transcriptional factor and its consensus sequence exists on the HER-2/neu promoter. To examine the role of PEA3 in HER-2/neu expression and cell transformation, we transfected PEA3 into the human breast and ovarian cancer cells that overexpress HER-2/neu and showed that PEA3 dramatically represses HER-2/neu transcription. PEA3 suppresses the oncogenic neu-mediated transformation in mouse fibroblast NIH 3T3 cells. Expression of PEA3 selectively blocks the growth of human cancer cells that overexpress HER-2/neu and inhibits their colony formation. It does not occur in the cancer cells expressing basal level of HER-2/neu. Further studies in the orthotopic ovarian cancer model demonstrated that expression of PEA3 preferentially inhibits growth and tumor development of human cancer cells that overexpress HER-2/neu, the tumor-bearing mice survived significantly longer if treated by injection of the PEA3-liposome complex intraperitoneally. Immunoblotting and immunohistochemical analysis of the tumor tissues indicated that PEA3 mediates the tumor suppression activity through targeting HER-2/neu-p185. Thus, PEA3 is a negative regulator of HER-2/neu gene expression and functions as a tumor suppressor gene in the HER-2/neu-overexpressing human cancer cells.^ The molecular mechanisms of PEA3 mediated transcriptional repression were investigated. PEA3 binds specifically at the PEA3 site on HER-2/neu promoter and this promoter-binding is required for the PEA3 mediated transcriptional repression. Mutation of the PEA3 binding site on HER-2/neu promoter causes decreased transcriptional activity, indicating that the PEA3 binding site is an enhancer-like element in the HER-2/neu-overexpressing cells. We therefore hypothesized that in the HER-2/neu-overexpressing cells, PEA3 competes with a transactivator for binding to the PEA3 site, preventing the putative factor from activating the transcription of HER-2/neu. This hypothesis was supported by the data which demonstrate that PEA3 competes with another nuclear protein for binding to the HER-2/neu promoter in vitro, and expression of a truncated protein which encodes the DNA binding domain of PEA3 is sufficient to repress HER-2/neu transcription in the HER-2/neu-overexpressing human cancer cells. ^

Functions of GCN5 and P /CAF acetyltransferases in mammalian development

Histone acetyltransferases are important chromatin modifiers that function as transcriptional co-activators. The identification of the transcriptional regulator GCN5 as the first nuclear histone acetyltransferase in yeast directly linked chromatin remodeling to transcriptional regulation. Although emerging evidence suggests that acetyltransferases participate in multiple cellular processes, their roles in mammalian development remain undefined. In this study, I have cloned and characterized the mouse homolog of GCN5 and a closely related protein P/CAF that interacts with p300/CBP. In contrast to yeast GCN5, but similar to P/CAF, mouse GCN5 possesses an additional N-terminal domain that confers the ability to acetylate nucleosomal histones. GCN5 and P/CAF exhibit identical substrate specificity and both interact with p300/CBP. Interestingly, expression levels of GCN5 and P/CAF display a complementary pattern in mouse embryos and in adult tissues, suggesting that they have distinct tissue or developmental stage specific roles. To define the in vivo function of GCN5 and P/CAF, I have generated mice that are nullizygous for GCN5 or P/CAF. P/CAF null mice are viable and fertile with no gross morphological defects, indicating that P/CAF is dispensable for development and p300/CBP function in vivo. In contrast, mice lacking GCN5 die between 10.5–11 days of gestation. GCN5 null mice are severely retarded but have anterior ectopic outgrowth. Molecular marker analyses reveal that early mesoderm is formed in GCN5 null mice but further differentiation into distinct mesodermal lineages is perturbed. While presomitic mesoderm and chodamesoderm are missing in GCN5 mutant mice, extraembryonic tissues and lateral mesoderm are unaffected. This is consistent with our finding that GCN5 expression is absent in the heart and extraembryonic tissues but is uniform throughout the rest of the embryo. Remarkably, GCN5 mutant mice exhibit an unusually high incidence of apoptosis in the embryonic ectoderm and mesoderm. Finally, mice doubly null for GCN5 and P/CAF die much earlier than mice harboring the GCN5 mutation alone, suggesting that P/CAF and GCN5 share some overlapping function during embryogenesis. This work is the first study to show that specific acetyltransferase is important for cell survival as well as mesoderm differentiation or maintenance during early mammalian development. ^

Functional analysis of SOX9 in chondrogenesis by targeted gene inactivation

Sox9 is a Sry-related HMG-domain containing transcription factor. Lines of evidence suggest that Sox9 has a potential role in skeletal development. During mouse development, Sox9 is predominantly expressed in all chondroprogenitors and differentiated chondrocytes, throughout the deposition of cartilage matrix. Mutations in one allele of SOX9 in humans result in campomelic dysplasia (CD), a skeletal dysplasia. syndrome characterized by the bowing of long bones. Moreover, Sox9 binds to and activates chondrocyte-specific enhancers in Col2a1 and Col11a2 genes. To further investigate the function of Sox9 in chondrogenesis, we analyzed chimeras derived from Sox9 heterozygous and homozygous null embryonic stem (ES) cells. In mouse chimeras, Sox9 −/− cells were excluded from all cartilages and did not express chondrocyte-specific genes. The segregation occurred during mesenchymal condensation. No cartilages developed in teratocarcinomas derived from Sox9 −/− ES cells. Mice heterozygous for the Sox9 mutation died neonatally and exhibited skeletal abnormalities resembling those of the CD patients. The Sox9 +/− mutants had a cleft palate and hypoplasia of scapula, pelvis and other skeletal structures derived by endochondral ossification. Bending of the radius, ulna and tibia cartilage was prominent at embryonic day 14.5 (E14.5). At E12.5 many pre-cartilaginous condensations were already defective. Advanced ossification was observed and the hypertrophic zone was enlarged in the growth plates, suggesting that Sox9 also regulates hypertrophic chondrocyte differentiation. Our results identify Sox9 as the first essential regulator of chondrocyte differentiation, which plays multiple roles in chondrogenesis. ^

Targeting a SWI/SNF-related chromatin remodeling complex to the β-globin promoter in erythroid cells

Chromatin remodeling complexes such as the SWI/SNF complex make DNA accessible to transcription factors by disrupting nucleosomes. However, it is not known how such complexes are targeted to the promoter. For example, a SWI/SNF1-like chromatin remodeling complex erythroid Krüppel-like factor (EKLF) coactivator-remodeling complex 1 (E-RC1) disrupts the nucleosomes over the human β-globin promoter in an EKLF-dependent manner. However, it is not known whether E-RC1 is targeted specifically to the β-globin promoter or whether E-RC1 is randomly targeted, but its activity is evident only at the β-globin promoter. Because E-RC1 cannot remodel chromatin over the β-globin promoter without EKLF in vitro, it has been proposed that SWI/SNF1-like complexes such as E-RC1 are targeted specifically to the promoter by selectively interacting with promoter-associated transcription factors such as EKLF. In this report, we test this hypothesis in the cellular context by using the ProteIN POsition Identification with Nuclease Tail (PIN*POINT) assay. We find that the Brahma-related gene (BRG) 1 and BRG1-associated factor (BAF) 170 subunits of E-RC1 are both recruited near the transcription initiation site of the β-globin promoter. On transiently transfected templates, both the locus control region and the EKLF-binding site are important for their recruitment to the β-globin promoter in mouse erythroleukemia cells. When the β-globin promoter was linked to the cytomegalovirus enhancer, the E-RC1 complex was not recruited, suggesting that recruitment of the E-RC1 complex is not a general property of enhancers.

Sustained in vivo cardiac protection by a rationally designed peptide that causes ɛ protein kinase C translocation

Brief periods of cardiac ischemia trigger protection from subsequent prolonged ischemia (preconditioning). ɛ Protein kinase C (ɛPKC) has been suggested to mediate preconditioning. Here, we describe an ɛPKC-selective agonist octapeptide, ψɛ receptor for activated C-kinase (ψɛRACK), derived from an ɛPKC sequence homologous to its anchoring protein, ɛRACK. Introduction of ψɛRACK into isolated cardiomyocytes, or its postnatal expression as a transgene in mouse hearts, increased ɛPKC translocation and caused cardio-protection from ischemia without any deleterious effects. Our data demonstrate that ɛPKC activation is required for protection from ischemic insult and suggest that small molecules that mimic this ɛPKC agonist octapeptide provide a powerful therapeutic approach to protect hearts at risk for ischemia.

Increased B-lymphopoiesis by interleukin 7 induces bone loss in mice with intact ovarian function: Similarity to estrogen deficiency

Estrogen deficiency caused by ovariectomy (OVX) results in a marked bone loss due to stimulated bone resorption by osteoclasts. During our investigations of the pathogenesis of bone loss in estrogen deficiency, we found that OVX selectively stimulates B-lymphopoiesis which results in marked accumulation of B220-positive pre-B cells in mouse bone marrow. To examine the possible correlation between stimulated B-lymphopoiesis and bone loss, 8-week-old female mice were treated with interleukin (IL) 7, which stimulates B-lymphopoiesis in bone marrow. We also examined bone mass in IL-7 receptor-knockout mice that exhibit marked suppression of B-lymphopoiesis in the bone marrow. The increased B-lymphopoiesis induced by IL-7 administration resulted in marked bone loss by stimulation of osteoclastic bone resorption in mice with intact ovarian function. The changes in both B-lymphopoiesis and bone mass in IL-7-treated female mice were similar to those in age-matched OVX mice. In contrast, the trabecular bone volume of the femur was greatly increased in both female and male IL-7 receptor-knockout mice when compared with the respective wild-type and heterozygous littermates. These results show that the perturbation of B-lymphopoiesis in the bone marrow is closely linked to the change in bone mass. We propose here that the increased B-lymphopoiesis due to estrogen deficiency is involved in the mechanism of stimulated bone resorption.

Impaired long-term potentiation induction in dentate gyrus of calretinin-deficient mice

Calretinin (Cr) is a Ca2+ binding protein present in various populations of neurons distributed in the central and peripheral nervous systems. We have generated Cr-deficient (Cr−/−) mice by gene targeting and have investigated the associated phenotype. Cr−/− mice were viable, and a large number of morphological, biochemical, and behavioral parameters were found unaffected. In the normal mouse hippocampus, Cr is expressed in a widely distributed subset of GABAergic interneurons and in hilar mossy cells of the dentate gyrus. Because both types of cells are part of local pathways innervating dentate granule cells and/or pyramidal neurons, we have explored in Cr−/− mice the synaptic transmission between the perforant pathway and granule cells and at the Schaffer commissural input to CA1 pyramidal neurons. Cr−/− mice showed no alteration in basal synaptic transmission, but long-term potentiation (LTP) was impaired in the dentate gyrus. Normal LTP could be restored in the presence of the GABAA receptor antagonist bicuculline, suggesting that in Cr−/− dentate gyrus an excess of γ-aminobutyric acid (GABA) release interferes with LTP induction. Synaptic transmission and LTP were normal in CA1 area, which contains only few Cr-positive GABAergic interneurons. Cr−/− mice performed normally in spatial memory task. These results suggest that expression of Cr contributes to the control of synaptic plasticity in mouse dentate gyrus by indirectly regulating the activity of GABAergic interneurons, and that Cr−/− mice represent a useful tool to understand the role of dentate LTP in learning and memory.

Detection of targeted GFP-Hox gene fusions during mouse embryogenesis

The ability to use a vital cell marker to study mouse embryogenesis will open new avenues of experimental research. Recently, the use of transgenic mice, containing multiple copies of the jellyfish gene encoding the green fluorescent protein (GFP), has begun to realize this potential. Here, we show that the fluorescent signals produced by single-copy, targeted GFP in-frame fusions with two different murine Hox genes, Hoxa1 and Hoxc13, are readily detectable by using confocal microscopy. Since Hoxa1 is expressed early and Hoxc13 is expressed late in mouse embryogenesis, this study shows that single-copy GFP gene fusions can be used through most of mouse embryogenesis. Previously, targeted lacZ gene fusions have been very useful for analyzing mouse mutants. Use of GFP gene fusions extends the benefits of targeted lacZ gene fusions by providing the additional utility of a vital marker. Our analysis of the Hoxc13GFPneo embryos reveals GFP expression in each of the sites expected from analysis of Hoxc13lacZneo embryos. Similarly, Hoxa1GFPneo expression was detected in all of the sites predicted from RNA in situ analysis. GFP expression in the foregut pocket of Hoxa1GFPneo embryos suggests a role for Hoxa1 in foregut-mediated differentiation of the cardiogenic mesoderm.

Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+: poly(adenosine-diphosphate-d-ribosyl)-acceptor ADP-d-ribosyltransferase, EC 2.4.2.30] is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents. To determine its biological function, we have inactivated both alleles by gene targeting in mice. Treatment of PARP−/− mice either by the alkylating agent N-methyl-N-nitrosourea (MNU) or by γ-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body γ-irradiation (8 Gy) mutant mice died rapidly from acute radiation toxicity to the small intestine. Mice-derived PARP−/− cells displayed a high sensitivity to MNU exposure: a G2/M arrest in mouse embryonic fibroblasts and a rapid apoptotic response and a p53 accumulation were observed in splenocytes. Altogether these results demonstrate that PARP is a survival factor playing an essential and positive role during DNA damage recovery.

Marked decrease of neuropeptide Y Y2 receptor binding sites in the hippocampus in murine prion disease

Using autoradiographic binding methodology with monoiodinated peptide YY together with the agonists neuropeptide Y (NPY) and NPY (13–36), as well as in situ hybridization with oligonucleotide probes complementary to the NPY Y2 receptor (Y2-R) mRNA, we have studied whether or not intracerebral prion inoculation affects Y2-Rs in male CD-1 mice. Monoiodinated peptide YY binding, mainly representing Y2-Rs, was down-regulated by 85% in the CA1 strata oriens and radiatum and by 50–65% in the CA3 stratum oriens 110–140 days postinoculation. In the CA3 stratum radiatum, where the mossy fibers from the dentate granule cells project, there was a significant decrease in PYY binding at 110–120 days. Y2-R mRNA, moderately expressed both in the CA1 and CA3 pyramidal cell layers and the granule cell layer in the dentate gyrus, showed a slight, but not significant, decrease in CA3 neurons 130 days postinoculation. The results indicate that the accumulation of the scrapie prion protein in the CA1–3 region strongly inhibits NPY binding at the Y2-Rs, which, however, is only marginally due to reduced Y2-R mRNA expression. The loss of the ability of NPY to bind to inhibitory Y2-Rs may cause dysfunction of hippocampal circuits and may contribute to the clinical symptoms in mouse scrapie.

Somatic hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: Possible role in antigen-driven reactions in the absence of germinal centers

The new antigen receptor (NAR) gene in the nurse shark diversifies extensively by somatic hypermutation. It is not known, however, whether NAR somatic hypermutation generates the primary repertoire (like in the sheep) or rather is used in antigen-driven immune responses. To address this issue, the sequences of NAR transmembrane (Tm) and secretory (Sec) forms, presumed to represent the primary and secondary repertoires, respectively, were examined from the peripheral blood lymphocytes of three adult nurse sharks. More than 40% of the Sec clones but fewer than 11% of Tm clones contained five mutations or more. Furthermore, more than 75% of the Tm clones had few or no mutations. Mutations in the Sec clones occurred mostly in the complementarity-determining regions (CDR) with a significant bias toward replacement substitutions in CDR1; in Tm clones there was no significant bias toward replacements and only a low level of targeting to the CDRs. Unlike the Tm clones where the replacement mutational pattern was similar to that seen for synonymous changes, Sec replacements displayed a distinct pattern of mutations. The types of mutations in NAR were similar to those found in mouse Ig genes rather than to the unusual pattern reported for shark and Xenopus Ig. Finally, an oligoclonal family of Sec clones revealed a striking trend toward acquisition of glutamic/aspartic acid, suggesting some degree of selection. These data strongly suggest that hypermutation of NAR does not generate the repertoire, but instead is involved in antigen-driven immune responses.