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Purpose: The purpose of this study was to assess the impact of anti-adenovirus neutralizing antibodies (AdNAbs) on the distribution, tolerability, and efficacy of intravenously administered oncolytic adenovirus. A translational model was developed to evaluate the impact of humoral immunity on intravenous administration of oncolytic adenovirus in humans., Experimental Design: Initially, severe combined immunodeficient (SCID)/beige mice were passively immunized with various amounts of human sera to establish a condition of preexisting humoral immunity similar to humans. A replication-deficient adenovirus encoding beta-galactosidase (rAd-betagal) was injected intravenously into these mice. An AdNAb titer that mitigated galactosidase transgene expression was determined. A xenograft tumor-bearing nude mouse model was developed to assess how a similar in vivo titer would impact the activity of 01/PEME, an oncolytic adenovirus, after intravenous administration., Results: In SCID/beige mice, there was a dose dependence between AdNAbs and galactosidase transgene expression; 90% of transgene expression was inhibited when the titer was 80. A similar titer reconstituted in the nude mice with human serum, as was done in the SCID/beige mice, did not abrogate the antitumor efficacy of the replicating adenovirus after intravenous administration. Viral DNA increased in tumors over time., Conclusions: In intravenous administration, preexisting AdNAb titer of 80 significantly attenuated the activity of a 2.5 x 10(12) particles per kilogram dose of nonreplicating adenovirus; the same titer had no affect on the activity of an equivalent dose of replicating adenovirus. Our results suggest that a majority of patients with preexisting adenovirus immunity would be candidates for intravenous administration of oncolytic adenovirus.

Central to the development of oncolytic virotherapies for cancer will be a better understanding of the parameters that influence the outcome of virotherapy to treat disseminated cancer by i.v. administration versus regional disease by local treatment. Intratumoral administration of 01/PEME, an oncolytic adenovirus, required approximately 1000-fold less dose than i.v. administration to induce similar tumor growth inhibition. Despite the short (<10 min) circulating half-life of the virus DNA, we could monitor virus distribution to the tumor site and observed virus replication by >1000-fold increase in virus DNA copies over time. There were doses of 01/PEME for which the virus DNA concentration in the tumor increased over time but did not result in antitumor efficacy. Oncolytic virus replication at a tumor site may not be a relevant indication of antitumor efficacy. Efficient distribution to the tumor site may be one of the most critical parameters for antitumor efficacy with oncolytic virotherapy.

Inducible synthesis and secretion of therapeutic proteins following gene transfer could be a viable strategy to deliver biopharmaceuticals that currently require parenteral administration. Evaluating the protein pharmacokinetics and biological responses generated by different delivery modalities will provide a better understanding of the advantages and disadvantages of each strategy. The interferon-alpha (IFN-alpha) family of proteins, used clinically for infectious and malignant diseases, has a short half-life, and IFN-alpha therapy requires frequent administration of the drug by injection. Subcutaneous xenograft tumors were inhibited by weekly administration of polyethylene glycol modified (PEGylated) IFN-alpha protein or by a single administration of an adenovirus constitutively expressing IFN-alpha (IACB). Both treatment modalities inhibited tumor growth in a dose-dependent manner, suggesting that increasing exposure to IFN-alpha could result in effective tumor control. A single adenovirus that encodes the components necessary for tetracycline induction (IADR) expressed IFN-alpha in a ligand-dependent manner. Adding doxycycline to the drinking water of mice treated intravenously with the inducible adenovirus IADR inhibited tumor growth by 85% compared with mice that were not given doxycycline. The correlation between serum IFN-alpha concentration and the degree of tumor growth inhibition did not depend on the delivery technology used. It is likely that it will be feasible to control expression of IFN-alpha by oral administration of small molecule drugs after gene delivery to induce therapeutic concentrations of proteins.

Gene delivery, with subsequent protein synthesis and secretion, in vivo has been proposed as an alternative way to deliver a therapeutic protein to the systemic circulation. Interferon-alpha (IFN) protein is effective in the treatment of viral and malignant diseases but has short serum half-life that requires frequent administration. An E1 region-deleted adenovirus vector encoding human IFN-alpha2b gene driven by the cytomegalovirus immediate early promoter (rAd-IFN) was generated to assess the serum concentration-time profiles of expressed IFN protein in animal models. Intravenous administration of rAd-IFN, normalized for body weight, resulted in dose-dependent serum IFN concentrations that persisted 8-40 days with similar concentration-time profiles in rats and rabbits. We sought to determine if serum concentration-time profiles in the rat and rabbit animal models would be predictive for a larger animal and would therefore be relevant models for potential dosing of human patients. Two chimpanzees (approximately 70 kg) dosed with rAd-IFN by intravenous administration normalized to body weight achieved serum IFN concentration-time profiles similar to those observed in rats and rabbits. The role of the immune response in limiting the persistence of transgene expression was highlighted by the persistence of serum IFN concentrations for over 200 days in beige/SCID immunodeficient mice. These studies suggest that serum concentration of secreted transgene products after gene delivery in small animal models may be highly predictive for larger species and will help define dosing strategies in human patients., ((c)2002 Elsevier Science (USA).)

Replicating adenoviruses may prove to be effective anticancer agents if they can be engineered to selectively destroy tumor cells. We have constructed a virus (01/PEME) containing a novel regulatory circuit in which p53-dependent expression of an antagonist of the E2F transcription factor inhibits viral replication in normal cells. In tumor cells, however, the combination of p53 pathway defects and deregulated E2F allows replication of 01/PEME at near wild-type levels. The re-engineered virus also showed significantly enhanced efficacy compared with extensively studied E1b-deleted viruses such as dl1520 in human xenograft tumor models.

A recombinant adenovirus expressing human interferon alpha2b driven by the cytomegalovirus promoter, IACB, was shown to produce and secrete biologically active protein in vitro and in vivo. Intravenous administration of IACB in Buffalo rats resulted in circulating levels of biologically active human interferon at 70,000 international units/mL for up to 15 days. Distribution of interferon protein after IACB administration was different from that seen with the subcutaneous delivery of interferon protein. Higher levels of interferon protein were observed in liver and spleen after IACB delivery compared to protein delivery. The antitumor efficacy of IACB, as measured by suppression of tumor growth, was tested in athymic nude mice bearing established human tumor xenografts from different types of human cancer. Subcutaneous tumors most responsive to the intratumoral administration of IACB ranked as U87MG (glioblastoma) and K562 (chronic myelogenous leukemia), followed by Hep 3B (hepatocellular carcinoma) and LN229 cells (glioblastoma). Intravenous administration of IACB in animals bearing U87MG or Hep 3B xenografts was also effective in suppressing tumor growth, although to a lesser extent than the intratumoral administration. IACB was also tested in a metastatic model in beige/SCID mice generated with H69 (small cell lung carcinoma) cells and was found to prolong survival in tumor-bearing animals. This suggested that interferon gene delivery can be effective in suppressing tumor growth in a wide variety of cells.

The oncolytic effect of adenoviruses may provide an efficient means to destroy tumor tissue if viruses could be developed with sufficient selectivity and efficacy. In this report we have characterized several adenoviruses, each with different mutations in the E1 region, for selective cytopathic effect in tumor cells in vitro and for their ability to inhibit tumor growth in vivo. Of the E1 mutants tested, we have identified one, E1Adl01/07, which preferentially induces cytopathic effects in a range of tumor cells versus primary cells. In addition, E1Adl01/07 significantly inhibited tumor growth and increased survival of mice in several models of human cancer. These results suggest that E1Adl01/07 might serve as an effective cancer therapeutic, combining both selectivity and efficacy.

Reports suggest a role of calpains in degradation of wild-type p53, which may regulate p53 induction of apoptosis. A calpain inhibitor, n-acetyl-leu-leu-norleucinal (calpain inhibitor 1), was assessed for ability to enhance p53-dependent apoptosis in human tumor cell lines with endogenous wild-type p53 and in altered p53 cell lines with the replacement of wild-type p53 by a recombinant adenovirus (rAd-p53). Calpain inhibitor 1 treatment resulted in increased levels of activated p53, increased p21 protein, and activation of caspases. Cell lines with wild-type, but not mutated or null, p53 status arrested in G0/G1 and were sensitive to calpain inhibitor-induced apoptosis. Regardless of endogenous p53 status, calpain inhibitor treatment combined with rAd-p53, but not empty vector virus, enhanced apoptosis in tumor cell lines. These results demonstrate p53-dependent apoptosis induced by a calpain inhibitor and further suggest a role for calpains in the regulation of p53 activity and induction of apoptotic pathways.

As a prelude to considering retinoblastoma (RB) gene therapy for cancer, a series of human tumor cell lines with either full-length, mutated, or undetectable RB protein were treated with recombinant adenovirus encoding RB (ACNRB). Both RB protein expression and the cytotoxic and antiproliferative effects of ACNRB treatment were evaluated. While the transgene expression of a reporter virus encoding the beta-galactosidase enzyme (rAd-beta-gal) varied among cell lines, the reintroduction and expression of the RB gene resulted in a pronounced inhibition of cellular proliferation in RB-altered cell lines. An antiproliferative response was observed with control adenovirus treatment in some cell lines. ACNRB treatment did not cause detectable cytotoxicity in either RB+ or RB-altered cells. Dose-dependent cytostasis was observed in RB- cell lines. In vivo tumor suppression was observed in a breast xenograft model subsequent to the treatment of established tumors with ACNRB. These data support a role for RB gene therapy of tumors with RB mutations and provide a basis for the further evaluation of ACNRB gene therapy of human cancer.

Mutated in multiple advanced cancers 1/phosphatase and tensin homologue (MMAC1/PTEN) is a novel tumor suppressor gene candidate located on chromosome 10 that is commonly mutated in human glioblastoma multiforme and several other cancer types. To evaluate the function of this gene as a tumor suppressor, we constructed a replication-defective adenovirus (MMCB) for efficient, transient transduction of MMAC1 into tumor cells. Infection of MMAC1-mutated U87MG glioblastoma cells with MMCB resulted in dose-dependent exogenous MMAC1 protein expression as detected by Western blotting of cell lysates. In vitro proliferation of U87MG cells was inhibited by MMCB in comparison to several control adenoviruses at equal viral doses, implying a specific effect of MMAC1 expression. Anchorage-independent growth in soft agar was also inhibited by MMCB compared to control adenovirus. Tumorigenicity in nude mice of transiently transduced mass cell cultures was then assessed. MMCB-infected U87MG cells were almost completely nontumorigenic compared to untreated and several control adenovirus-treated cells at equal viral doses. These data support an in vivo tumor suppression activity of MMAC1/PTEN and suggest that in vivo gene transfer with this recombinant adenoviral vector has a potential use in cancer gene therapy.

The development of neoplasia frequently involves inactivation of the p53 and retinoblastoma (Rb) tumor suppressor pathways and disruption of cell cycle checkpoints that monitor the integrity of replication and cell division. The human papillomavirus type 16 (HPV-16) oncoproteins, E6 and E7, have been shown to bind p53 and Rb, respectively. To further delineate the mechanisms by which E6 and E7 affect cell cycle control, we examined various aspects of the cell cycle machinery. The low-risk HPV-6 E6 and E7 proteins did not cause any significant change in the levels of cell cycle proteins analyzed. HPV-16 E6 resulted in very low levels of p53 and p21 and globally elevated cyclin-dependent kinase (CDK) activity. In contrast, HPV-16 E7 had a profound effect on several aspects of the cell cycle machinery. A number of cyclins and CDKs were elevated, and despite the elevation of the levels of at least two CDK inhibitors, p21 and p16, CDK activity was globally increased. Most strikingly, cyclin E expression was deregulated both transcriptionally and posttranscriptionally and persisted at high levels in S and G2/M. Transit through G1 was shortened by the premature activation of cyclin E-associated kinase activity. Elevation of cyclin E levels required both the CR1 and CR2 domains of E7. These data suggest that cyclin E may be a critical target of HPV-16 E7 in the disruption of G1/S cell cycle progression and that the ability of E7 to regulate cyclin E involves activities in addition to the release of E2F.

Purpose: To develop a checkpoint-based strategy for preferential radiosensitization of human tumors with deficient and/or mutant p53., Methods and Materials: A549 human lung adenocarcinoma cell lines differing in their expression of the p53 tumor suppressor gene were produced by transduction with the E6 oncogene from human papilloma virus type 16. The cells expressing E6 (E6+) lack a G1 arrest in response to ionizing radiation, are deficient in p53 and p21 expression, and exhibit a fivefold greater clonogenic survival following 10 Gy radiation., Results: Postirradiation incubation with millimolar concentrations of the methylxanthine pentoxifylline (PTX) results in preferential radiosensitization of the E6+ cells compared to the LXSN+ vector transduced controls. There is a threefold sensitization of the LXSN+ cells and a 15-fold sensitization of the E6+ cells, which results in equal clonogenic survival of the two lines. Flow cytometry reveals PTX abrogation of the radiation induced G2 arrest for both cell lines. PTX also prolongs G1 transit for both cell lines. Preliminary results are presented using a novel methylxanthine, lisofylline (LSF), which has similar cell cycle effects on G1 and G2 and achieves differential radiosensitization at micromolar concentrations that are sustainable in humans., Conclusion: This checkpoint-based strategy is a promising approach for achieving preferential radiosensitization of p53- tumors relative to p53+ normal tissues.

Cells arrest in the G1 or G0 phase of the cell cycle in response to a variety of negative growth signals that induce arrest by different molecular pathways. The ability of human papillomavirus (HPV) oncogenes to bypass these signals and allow cells to progress into the S phase probably contributes to the neoplastic potential of the virus. The E7 protein of HPV-16 was able to disrupt the response of epithelial cells to three different negative growth arrest signals: quiescence imposed upon suprabasal epithelial cells, G1 arrest induced by DNA damage, and inhibition of DNA synthesis caused by treatment with transforming growth factor beta. The same set of mutated E7 proteins was able to abrogate all three growth arrest signals. Mutant proteins that failed to abrogate growth arrest signals were transformation deficient and included E7 proteins that bound retinoblastoma protein in vitro. In contrast, HPV-16 E6 was able to bypass only DNA damage-induced G1 arrest, not suprabasal quiescence or transforming growth factor beta-induced arrest. The E6 and E7 proteins from the low-risk virus HPV-6 were not able to bypass any of the growth arrest signals.

Many tumor types have p53 and/or RB mutations, and it is unclear what role the mutations of these tumor suppressor genes have on the efficacy of chemotherapeutic agents. The effect of p53 and RB inactivation on sensitivity to chemotherapeutic drugs was examined using a model system in which p53 or RB was inactivated in normal human foreskin fibroblasts (HFFs) by acute expression of human papillomavirus (HPV) 16E6 or 16E7. Cytotoxicity assays showed that HFFs expressing HPV 16E6 were 6- to 9-fold more sensitive to the DNA crosslinkers cisplatin and carboplatin and 7.8- to 11.5-fold more sensitive to the tubulin polymerizing agent paclitaxel than were LXSN-expressing cells. Analysis of mouse embryonal fibroblasts lacking p53 (p53-/-) compared with mouse embryonal fibroblasts homozygous (p53+/+) and heterozygous (p53+/-) for wild-type p53 confirmed the role of p53 in the enhanced sensitivity to cisplatin. Treatment with the alkylating agents melphalan and nitrogen mustard resulted in 3.8- to 7.3-fold greater sensitivity in HPV 16E6- or 16E7-expressing cells compared with LXSN-expressing cells. Enhanced sensitivity to cisplatin in cells lacking p53 function was explored by examination of its effects on cell cycle progression after exposure. When treated with cisplatin, HFFs expressing 16E6 showed delayed progression through S phase relative to HFFs expressing LXSN. The delay in S phase progression was coincident with the induction of p53 protein levels in LXSN-containing HFFs, suggesting a role for p53 in DNA repair of cisplatin-induced damage. These results indicate that the inactivation of p53 in the absence of other genetic alterations leads to enhanced sensitivity to multiple chemotherapeutic agents rather than to increased resistance.

The anticancer agent paclitaxel (Taxol) stabilizes tubulin polymerization resulting in arrest in mitosis and apoptotic cell death. Normal human fibroblasts depleted of functional p53 by SV40 T antigen or HPV-16 E6, and primary embryo fibroblasts from p53 null mice showed seven- to ninefold increased cytotoxicity by paclitaxel. Reduced levels of p53 correlated with increased G2/M phase arrest, micronucleation, and p53-independent paclitaxel-induced apoptosis. Surviving cells with intact p53 progressed through mitosis and transiently accumulated in the subsequent G1 phase, coincident with increased p53 and p21cip1,waf1 protein levels. These results are in contrast to studies linking p53 loss with resistance to DNA damaging anticancer agents.

Sequence differences in the noncoding region of the human papillomavirus type 16 (HPV-16) genome were displayed using single-stranded conformational polymorphism (SSCP) analysis of polymerase chain reaction (PCR)-amplified material. Two variants accounted for 50%-70% of all HPV-16 variants from 3 cohorts in Seattle. Seventy subjects who were repeatedly HPV-16 DNA-positive over 2-8 4-monthly visits showed an identical SSCP pattern at every visit. Only 10%-20% of the specimens showed evidence of infection by multiple variants when assessed by SSCP. However, cloning and sequencing of the PCR products revealed a substantially higher proportion of specimens with > 1 variant. Sequencing many clones from each specimen confirmed that 1 major variant seemed to predominate over time, whereas minor variants appeared more transient. These results suggest that HPV-16 establishes a persistent infection in which a single variant predominates: coinfection with addition HPV-16 variants results in a minor population of HPV-16 genomes.

We have examined the effect of abrogation of the G2 checkpoint on the radiosensitivity of G1 checkpoint-proficient and G1 checkpoint-deficient cells. A549 human lung adenocarcinoma cells were transduced with the E6 oncogene of the human papillomavirus type 16 to eliminate their radiation-induced G1 arrest. These E6+ cells exhibited a dose-dependent increase in radiation resistance compared to control A549 cells transduced with the vector alone. Treatment (96 h) with 2 mM caffeine resulted in an abrogation of the cellular G2 checkpoint in both E6+ and control cells and a differential radiosensitizing effect on the two cell lines such that the E6+ clones and the vector controls became equally radiosensitive. These data show that human tumors which are radioresistant due to the loss of the p53-mediated G1 checkpoint can be made radiosensitive by abrogation of the G2 checkpoint. The implications of these results for cancer therapy are discussed.

Functional p53 protein is associated with the ability of cells to arrest in G1 after DNA damage. The E6 protein of cancer-associated human papillomavirus type 16 (HPV-16) binds to p53 and targets its degradation through the ubiquitin pathway. To determine whether the ability of E6 to interact with p53 leads to a disruption of cell cycle control, mutated E6 proteins were tested for p53 binding and p53 degradation targeting in vitro, the ability to reduce intracellular p53 levels in vivo, and the ability to abrogate actinomycin D-induced growth arrest in human keratinocytes. Mutations scattered throughout the amino terminus, either zinc finger or the central region but not the carboxy terminus, severely reduced the ability of E6 to interact with p53. Expression of HPV-16 E6 or mutated E6 proteins that bound and targeted p53 for degradation in vitro sharply reduced the level of intracellular p53 induced by actinomycin D in human keratinocytes. A perfect correlation between the ability of E6 proteins to reduce the level of intracellular p53 and their ability to block actinomycin D-induced cellular growth arrest was observed. These results suggest that interaction with p53 is important for the ability of HPV E6 proteins to circumvent growth arrest.

Cellular tumor suppressors p53 and Rb play an important role in controlling cell proliferation. Inactivation of these tumor suppressor proteins can occur by gene mutation or by association with oncoproteins from the small DNA tumor viruses. One function of p53 is in regulating cell cycle check-point control after DNA damage. To dissect the pathways by which p53 and Rb may act, the E6 and E7 oncogenes of human papillomavirus (HPV) types 6 and 16 were introduced into primary human epithelial cells by retroviral transfer vector, and cells were assayed for growth arrest after DNA damage induced by actinomycin D. The E6 or E7 oncogenes from the low-risk HPV6 had no affect on growth arrest, p53 protein levels increased, Rb protein levels decreased, and Rb was predominantly in the hypophosphorylated state similar to vector-infected cells. Either the E6 or the E7 oncogene from the high-risk HPV16 abrogated growth arrest. Cells expressing HPV16 E6 (16E6) were severely reduced in p53 protein levels that did not increase detectably after DNA damage, Rb protein levels did not decrease, and hyperphosphorylated Rb was present. After DNA damage in cells expressing 16E7 p53 levels increased, and Rb protein levels decreased; however, Rb was predominantly in the hyperphosphorylated state. Even though p53 protein levels increased in response to DNA damage in cells expressing 16E7, G1 growth arrest was bypassed. This suggests that the circuitry controlling the growth arrest signal after DNA damage may be interconnected between the p53 and Rb pathways.

The role tumor suppressors p53 and retinoblastoma (RB) play in the transformation process has become central to understanding the pathogenesis of DNA tumor viruses. The two oncoproteins of human papillomavirus (HPV)-16, E6 and E7, bind to p53 and RB, respectively, thus inactivating the function of these tumor suppressor genes. Immortalization of primary human foreskin epithelial cells by HPV requires expression of the E7 protein, and the E6 protein greatly enhances the immortalization frequency. Two of three cell lines immortalized by the HPV-16 E7 oncoprotein expressed wild-type p53 and only one of the three cell lines had acquired a p53 mutation and loss of heterozygosity at 17p during the immortalization process. All three E7-immortalized lines contained higher steady-state levels of p53 protein. Mutation of the p53 gene is not required for immortalization in the absence of the HPV-16 E6 inactivation of the p53 protein, and 16E7 expression leads to the stabilization of wild-type p53.

Previous studies have shown that the E7 gene of human papillomavirus (HPV) type 16 or 18 alone was sufficient for immortalization of human foreskin epithelial cells (HFE) and that the efficiency was increased in cooperation with the respective E6 gene, whereas the HPV6 E6 or E7 gene was not active in HFE. To detect weak immortalizing activities of the HPV6 genes, cells were infected with recombinant retroviruses containing HPV genes, alone and in homologous and heterologous combinations. The HPV6 genes, alone or together (HPV6 E6 plus HPV6 E7), were not able to immortalize cells. However the HPV6 E6 gene, in concert with HPV16 E7, increased the frequency of immortalization threefold over that obtained with HPV16 E7 alone. Interestingly, 6 of 20 clones containing the HPV16 E6 gene and the HPV6 E7 gene were immortalized, whereas neither gene alone was sufficient. Thus, the HPV6 E6 and E7 genes have weak immortalizing activities which can be detected in cooperation with the more active transforming genes of HPV16. Acute expression of the HPV6 and HPV16 E6 and E7 genes revealed that only HPV16 E7 was able to stimulate the proliferation of cells in organotypic culture, resulting in increased expression of the proliferative cell nuclear antigen and the formation of a disorganized epithelial layer. Additionally, combinations of genes that immortalized HFE cells (HPV16 E6 plus HPV16 E7, HPV16 E6 plus HPV6 E7, and HPV6 E6 plus HPV16 E7) also stimulated proliferation.

The contribution of the E6 and E7 open reading frames of human papillomavirus type 6b (HPV6b) and HPV16 to immortalization of human keratinocytes was evaluated by using amphotropic recombinant retroviruses. The HPV16 E7 gene could immortalize primary human keratinocytes without the cooperation of the viral E6 gene; however, E6 was able to contribute significantly to the efficiency of the E7 immortalizing function. Infection of HFE cells with retroviruses carrying the 16E6, 6bE6, or 6bE6E7 open reading frame did not result in immortalization.

Determination of the DNA sequence of a cluster of repetitive elements located 3' to the rabbit beta-1 globin gene shows that previously identified B, E, and D repeats are linked to form a long repeat. The B and E regions are homologous to the L1 long interspersed repeats of primates and rodents. Thus L1 repeats are widely dispersed in several different mammalian orders. The sequence similarity among L1 repeats from three species is limited to a long segment that contains extensive open reading frames. This conserved region is followed by a highly divergent segment that corresponds to a 3' untranslated region. The conservation of sequences in the open-reading-frame region coupled with the divergence of the untranslated region supports the hypothesis that the L1 repeats are derived from transcripts of a functional gene that encodes a protein.

Rabbit colonic loops were exposed to purified enterotoxin from Clostridium perfringens type A and perfused for analysis of the effects of the enterotoxin on transport and surface structure of the colon. The colon showed no apparent response to the enterotoxin in the transport of fluid and electrolytes as compared with untreated animals. There was a significant increase in levels of luminal fluid protein, corresponding to an observed increase in secretion of mucus. Histologic sections showed no epithelial damage, but scanning electron microscopy showed an increase in the number of mucous fields over the value in control animals. The colon showed little or no overt response to enterotoxin treatment, but isolated colonic epithelial cells bound 125I-labeled enterotoxin at levels even greater than those observed in cells from the ileum, where the enterotoxin has considerable biologic activity. Thus, although binding of enterotoxin is necessary for its biologic activity, biologic activity does not necessarily follow binding to specific receptors.

The L1 family of long interspersed repetitive DNA in the rabbit genome (L1Oc) has been studied by determining the sequence of the five L1 repeats in the rabbit beta-like globin gene cluster and by hybridization analysis of other L1 repeats in the genome. L1Oc repeats have a common 3' end that terminates in a poly A addition signal and an A-rich tract, but individual repeats have different 5' ends, indicating a polar truncation from the 5' end during their synthesis or propagation. As a result of the polar truncations, the 5' end of L1Oc is present in about 11,000 copies per haploid genome, whereas the 3' end is present in at least 66,000 copies per haploid genome. One type of L1Oc repeat has internal direct repeats of 78 bp in the 3' untranslated region, whereas other L1Oc repeats have only one copy of this sequence. The longest repeat sequenced, L1Oc5, is 6.5 kb long, and genomic blot-hybridization data using probes from the 5' end of L1Oc5 indicate that a full length L1Oc repeat is about 7.5 kb long, extending about 1 kb 5' to the sequenced region. The L1Oc5 sequence has long open reading frames (ORFs) that correspond to ORF-1 and ORF-2 described in the mouse L1 sequence. In contrast to the overlapping reading frames seen for mouse L1, ORF-1 and ORF-2 are in the same reading frame in rabbit and human L1s, resulting in a discistronic structure. The region between the likely stop codon for ORF-1 and the proposed start codon for ORF-2 is not conserved in interspecies comparisons, which is further evidence that this short region does not encode part of a protein. ORF-1 appears to be a hybrid of sequences, of which the 3' half is unique to and conserved in mammalian L1 repeats. The 5' half of ORF-1 is not conserved between mammalian L1 repeats, but this segment of L1Oc is related significantly to type II cytoskeletal keratin.

The nucleotide sequence of the entire beta-like globin gene cluster of rabbits has been determined. This sequence of a continuous stretch of 44.5 x 10(3) base-pairs (bp) starts about 6 x 10(3) bp upstream from epsilon (the 5'-most gene) and ends about 12 x 10(3) bp downstream from beta (the 3'-most gene). Analysis of the sequence reveals that: (1) the sequence is relatively A + T rich (about 60%); (2) regions with high G + C content are associated with OcC repeats, a short interspersed repeated DNA in rabbits; (3) the distribution of polypurines, polypyrimidines and alternating purine/pyrimidine tracts is not random within the cluster; (4) most open reading frames are associated with known globin coding regions, OcC repeats or long interspersed repeats (L1 repeats); (5) the most prominent open reading frames are found in the L1 repeats; (6) different strand asymmetries in base composition are associated with embyronic and adult genes as well as the tandem L1 repeats at the 3' end of the cluster; and (7) essentially all the repeats appear to have been inserted by a transposon mechanism. A comparison of the sequence with itself by a dot-plot analysis has revealed nine new members of the OcC family of repeats in addition to the six previously reported. The OcC repeats tend to be clustered, particularly in the epsilon-gamma and gamma-psi delta intergenic regions. Dot-plot comparisons between the rabbit and the human clusters have revealed extensive sequence matches. Homology starts about 6 x 10(3) bp 5' to epsilon or as far upstream as the rabbit sequence is available. It continues throughout the entire cluster and stops about 0.7 x 10(3) bp 3' to beta, at which point several repeats have inserted in both rabbits and humans. Throughout the gene cluster, the homology is interrupted mainly by insertions or deletions in either the rabbit or the human genome. Almost all of the insertions are of known short or long repeated DNAs. The positions of the insertions are different in the two gene clusters, which indicates that both short and long repeats have been transposing throughout the genome for the time since the mammalian radiation. An alignment of rabbit and human sequences allows the calculation of the substitution rate around epsilon. Sequences far removed from the gene are evolving at a rate equivalent to the pseudogene rate, although some short regions show an apparently higher rate.(ABSTRACT TRUNCATED AT 400 WORDS)

The general pattern of sequence matches between the beta-like globin gene clusters of rabbits and humans are summarized in Fig. 7. The regions of matching sequences are shaded, and it can be seen that the matches extend from one end of the gene cluster to the other. This provides very strong evidence that the ancestral species had a gene cluster containing the parents to all the contemporary beta-like globin genes in the same arrangement that we observe today. Much of the intergenic DNA has been diverging at a rate consistent with neutral drift, but smaller regions can be detected that are diverging more slowly and which are good candidates for functional sequences. The comparisons between this same gene cluster in mouse and humans show many fewer matches in the intergenic regions (Shehee et al., 1989), indicating either an earlier split between rodents and primates or a faster rate of divergence in rodents. However, this more divergent sequence may prove particularly valuable in a search for functional sequences, especially in a three-way alignment between the sequenced gene clusters. Every repetitive element in homologous segments of the rabbit and human beta-like globin gene clusters interrupts the homology; no repeat is in the same position in both species. Hence all the repeats have been inserted into the gene clusters after the divergence between lagomorphs and primates. This is true even for the L1 repeats, which are very similar between species in their ORF regions. This pattern of interspersion of repeats in long orthologous regions shows that many members of the LINE and SINE families are recent additions to the genome, and that these repeats are in fact transposable elements. It is easy to imagine negative and neutral effects of the expansion and transpositions of these repeat families, but some positive effect has not been ruled out. One of the intriguing inferences from the observations about repeats is that the ancestral gene cluster may not have contained repetitive elements. If it did, then those repeats have been completely replaced by different repeats independently in lagomorphs and primates.