Increased spontaneous mutation frequency in human cells expressing the phage PBS2-encoded inhibitor of uracil-DNA glycosylase.

The Ugi protein inhibitor of uracil-DNA glycosylase encoded by bacteriophage PBS2 inactivates human uracil-DNA glycosylases (UDG) by forming a tight enzyme:inhibitor complex. To create human cells that are impaired for UDG activity, the human glioma U251 cell line was engineered to produce active Ugi protein. In vitro assays of crude cell extracts from several Ugi-expressing clonal lines showed UDG inactivation under standard assay conditions as compared to control cells, and four of these UDG defective cell lines were characterized for their ability to conduct in vivo uracil-DNA repair. Whereas transfected plasmid DNA containing either a U:G mispair or U:A base pairs was efficiently repaired in the control lines, uracil-DNA repair was not evident in the lines producing Ugi. Experiments using a shuttle vector to detect mutations in a target gene showed that Ugi-expressing cells exhibited a 3-fold higher overall spontaneous mutation frequency compared to control cells, due to increased C:G to T:A base pair substitutions. The growth rate and cell cycle distribution of Ugi-expressing cells did not differ appreciably from their parental cell counterpart. Further in vitro examination revealed that a thymine DNA glycosylase (TDG) previously shown to mediate Ugi-insensitive excision of uracil bases from DNA was not detected in the parental U251 cells. However, a Ugi-insensitive UDG activity of unknown origin that recognizes U:G mispairs and to a lesser extent U:A base pairs in duplex DNA, but which was inactive toward uracil residues in single-stranded DNA, was detected under assay conditions previously shown to be efficient for detecting TDG.

erbB-2 overexpression in human mammary epithelial cells confers growth factor independence.

Previously, we demonstrated that human breast cancer cells with progressively elevated levels of constitutively tyrosine phosphorylated erbB-2 are independent of growth factors required by normal human mammary epithelial (HME) cells for proliferation in serum-free medium. To determine whether erbB-2 overexpression alone is sufficient to confer the growth factor-independence phenotype in HME cells, the spontaneously immortalized MCF-10A cell line and the HPV-16-immortalized H16N2 cell line were infected with the bicistronic retroviral vector pTPerbB-2 and tested for their ability to grow in the absence of specific factors. Selection of infected cells in G418-containing medium resulted in moderate levels of erbB-2 overexpression in approximately 40% of cells. The subpopulation of erbB-2 overexpressing cells could be selected for by culturing the cells in medium devoid of insulin. When MCF-10A or H16N2 cells were infected with pTPerbB-2 and directly selected in growth factor-deficient medium over long periods of time, populations of both cell lines emerged that expressed levels of erbB-2 protein equivalent to levels expressed by breast cancer cells with an erbB-2 gene amplification. Furthermore, overexpressed p185(erbB-2) was constitutively tyrosine phosphorylated in these cells. The levels of tyrosine phosphorylated p185(erbB-2) differed in the two recipient lines, with H16N2-erbB-2 cells having higher levels of activated receptor than MCF-10AerbB-2 cells. Furthermore, only the H16N2-erbB-2 cells were independent of both insulin and epidermal growth factor for growth in serum-free medium. Overexpression of erbB-2 also resulted in progressively increasing levels of tyrosine-phorphorylated erbB-3, without any significant changes in p180(erbB-3) levels. These studies demonstrate a direct relationship between the level of expression and activation of p185(erbB-2) and the requirements of HME cells for insulin-like and epidermal growth factor-like growth factors. The results also suggest that genetic alterations present in breast cancer cells, or mediated by HPV-16-induced alterations in pRb and p53, can influence the expression level and activation status of erbB-2 as well as erbB-3 and, in turn, their degree of growth factor independence.

Patterns of failure following high-dose 3-D conformal radiotherapy for high-grade astrocytomas: a quantitative dosimetric study.

PURPOSE: To analyze the failure patterns for patients with high-grade astrocytomas treated with high-dose conformal radiotherapy (CRT) using a quantitative technique to calculate the dose received by the CT- or MR-defined recurrence volume and to assess whether the final target volume margin used in the present dose escalation study requires redefinition before further escalation. METHODS AND MATERIALS: Between 4/89 and 10/95, 71 patients with high-grade supratentorial astrocytomas were entered in a phase I/II dose escalation study using 3-D treatment planning and conformal radiotherapy. All patients were treated to either 70 or 80 Gy in conventional daily fractions of 1.8-2.0 Gy. The clinical and planning target volumes (CTV, PTV) consisted of successively smaller volumes with the final PTV defined as the enhancing lesion plus 0.5 cm margin. As of 10/95, 47 patients have CT or MR evidence of disease recurrence/progression. Of the 47 patients, 36 scans obtained at the time of recurrence were entered into the 3-D radiation therapy treatment planning system. After definition of the recurrent tumor volumes, the recurrence scan dataset was registered with the pretreatment CT dataset so that the actual dose received by the recurrent tumor volumes during treatment could be accurately calculated and then analyzed dosimetrically using dose-volume histograms. Recurrences were divided into several categories: 1) "central," in which 95% or more of the recurrent tumor volume (Vrecur) was within D95, the region treated to high dose (95% of the prescription dose); 2) "in-field," in which 80% or more of Vrecur was within the D95 isodose surface; 3) "marginal," when between 20 and 80% of Vrecur was inside the D95 surface; 4) "outside," in which less than 20% of Vrecur was inside the D95 surface. RESULTS: In 29 of 36 patients, a solitary lesion was seen on recurrence scans. Of the 29 solitary recurrences, 26 were central, 3 were marginal, and none were outside. Multiple recurrent lesions were seen in seven patients: three patients had multiple central and/or in-field lesions only, three patients had central and/or in-field lesions with additional small marginal or outside lesions, and one patent had 6 outside and one central lesion. Since total recurrence volume was used in the final analysis, 6 of the 7 patients with multiple recurrent lesions were classified into centra/in-field category. CONCLUSION: Analysis of the 36 evaluable patients has shown that 32 of 36 patients (89%) failed with central or in-field recurrences, 3/36 (8%) had a significant marginal component to the recurrence, whereas only 1/36 (3%) could be clearly labeled as failing mainly outside the high-dose region. Seven patients had multiple recurrences, but only 1 of 7 had large-volume recurrences outside the high-dose region. This study shows that the great majority of patient recurrences that occur after high-dose (70 or 80 Gy) conformal irradiation are centrally located: only 1/36 patients (with 7 recurrent lesions) had more than 50% of the recurrence volume outside the region previously treated to high dose. Further dose escalation to 90 Gy (and beyond) thus seems reasonable, based on the same target volume definition criteria

Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells.

PURPOSE: We have reported previously that the expression of E. coli dUTPase (dutE) can protect HT29 cells from 5-fluorodeoxyuridine (FdUrd)-induced DNA fragmentation and cytotoxicity. In the study reported here, we further characterized the ability of dutE expression in one HT29 clone, dutE7, to alter the effects of treatment with FdUrd and other thymidylate synthase (TS) inhibitors. In addition, we developed two HuTu80 dutE-expressing clones using a pLNCX-dutE retroviral construct and tested their sensitivity to FdUrd-induced DNA fragmentation and cytotoxicity. METHODS: Both a dutE retroviral expression system and a dutE antibody were developed to facilitate the generation and screening of dutE-expressing clones. HT29 and HuTu80 clones expressing dutE were tested for drug-induced DNA damage with either alkaline elution or pulsed field gel electrophoresis and drug-induced loss of clonogenicity. RESULTS: Following a 24-h treatment with 100 microM CB3717 or 500 nM methotrexate (MTX), dutE7 cells were significantly less sensitive to drug-induced loss of clonogenicity than con3 cells. DutE7 cells were also resistant to CB3717-induced DNA fragmentation at 24 h. However, following a 48-h treatment with CB3717 or MTX there was no difference in survival between con3 and dutE7 cells, even though DNA damage was still greatly attenuated in the dutE7 cell line. In addition, expression of dutE in two HuTu80 clones, 80 C and 80 K, did not protect these cells from FdUrd-induced DNA damage or cytotoxicity. CONCLUSIONS: We conclude that the role of uracil misincorporation and subsequent DNA damage in cytotoxicity induced by TS inhibitors, in HT29 cells, is time dependent, and that cytotoxicity caused by long-term exposure to these drugs is largely independent of resultant DNA damage, in this cell line. The inability of dutE to protect HuTu80 cells from FdUrd further suggests that the significance of uracil misincorporation resulting from TS inhibition varies among cell lines.

A brain tumor dose escalation protocol based on effective dose equivalence to prior experience.

PURPOSE: The current study describes the design of a dose escalation protocol for conformal irradiation of primary brain tumors that preserves the safe experience of a previous, sequential dose escalation scheme while enabling the delivery of substantially higher effective doses to a central target volume. METHODS AND MATERIALS: Normalized isoeffective composite dose distributions were formed for 20 patients treated on the original protocol (which specified three progressively smaller planning target volumes [PTVs]) using the linear quadratic model (here corrected to equivalent 2 Gy fractions using alpha/beta=10 Gy). These distributions were investigated and a new protocol was designed to preserve a similar level of efficacy and lack of toxicity for the outer volumes, but allowing a higher dose to the inner PTV. Treatment plans were then investigated to determine if the objectives of the new protocol were achievable. In particular, plans that simultaneously achieved all biological treatment planning objectives (all fields treated each day) were investigated. Finally, the success of the protocol design was demonstrated by analysis of the effective dose distributions of 10 patients treated using the new protocol. RESULTS: The composite normalized isoeffective minimum doses to the outer PTVs (PTV3 and PTV2) in the original protocol were close to 60 Gy and 75 Gy, respectively, and these values are specified as the minimum doses to those volumes for the new protocol. Homogeneity requirements to maintain equivalence for the outer target volume domains are: not more than 25% of [PTV3 exclusive of PTV2] >75 Gy; and not more than 50% of [PTV2 exclusive of PTV1] >85 Gy. Treatment plans using multiple noncoplanar arrangements of beams and static intensity modulation treat all volumes at each session. DVHs of the normalized isoeffective dose distributions reveal the equivalence of the new protocol plans to the sequential plans in the previous protocol as well as the ability to achieve a higher dose of 90 Gy to the isocenter of PTV1 (+/-5% homogeneity required). CONCLUSION: The ability to incorporate past experience through use of the linear quadratic model in the design of a new dose escalation protocol is demonstrated.

Novel mechanisms of E2F induction by BK virus large-T antigen: requirement of both the pRb-binding and the J domains.

E2F activity is regulated in part by the retinoblastoma family of tumor suppressor proteins. Viral oncoproteins, such as simian virus 40 (SV40) large-T antigen (TAg), adenovirus E1A, and human papillomavirus E7, can disrupt the regulation of cellular proliferation by binding to pRb family members and dissociating E2F-pRb family protein complexes. BK virus (BKV), which infects a large percentage of the human population and has been associated with a variety of human tumors, encodes a TAg homologous to SV40 TAg. It has been shown that BKV TAg, when expressed at low levels, does not detectably bind to pRb family members, yet it induces a serum-independent phenotype and causes a decrease in the overall levels of pRb family proteins. The experiments presented in this report show that, despite the lack of TAg-pRb interactions, BKV TAg can induce transcriptionally active E2F and that this induction does in fact require an intact pRb-binding domain as well as an intact J domain. In addition, E2F-pRb family member complexes can be detected in both BKV and SV40 TAg-expressing cells. These results suggest the presence of alternate cellular mechanisms for the release of E2F in addition to the well-established model for TAg-pRb interactions. These results also emphasize a role for BKV TAg in the deregulation of cellular proliferation, which may ultimately contribute to neoplasia.

Mouse mammary tumor virus/v-Ha-ras transgene-induced mammary tumors exhibit strain-specific allelic loss on mouse chromosome 4.

Hybrid mice carrying oncogenic transgenes afford powerful systems for investigating loss of heterozygosity (LOH) in tumors. Here, we apply this approach to a neoplasm of key importance in human medicine: mammary carcinoma. We performed a whole genome search for LOH using the mouse mammary tumor virus/v-Ha-ras mammary carcinoma model in female (FVB/N x Mus musculus castaneus)F1 mice. Mammary tumors developed as expected, as well as a few tumors of a second type (uterine leiomyosarcoma) not previously associated with this transgene. Genotyping of 94 anatomically independent tumors revealed high-frequency LOH ( approximately 38%) for markers on chromosome 4. A marked allelic bias was observed, with M. musculus castaneus alleles almost exclusively being lost. No evidence of genomic imprinting effects was noted. These data point to the presence of a tumor suppressor gene(s) on mouse chromosome 4 involved in mammary carcinogenesis induced by mutant H-ras expression, and for which a significant functional difference may exist between the M. musculus castaneus and FVB/N alleles. Provisional subchromosomal localization of this gene, designated Loh-3, can be made to a distal segment having syntenic correspondence to human chromosome 1p; LOH in this latter region is observed in several human malignancies, including breast cancers. Evidence was also obtained for a possible second locus associated with LOH with less marked allele bias on proximal chromosome 4.

Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway. I: DNA base and nucleolytic specificity.

Cells of Bacillus subtilis can enter a natural physiological state, termed competence, that is permissive for uptake of DNA from the surrounding medium. In the B. subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, followed by intracellular processing that may ultimately lead to productive infection. Previous investigations have shown that transfecting DNA is usually far less infectious (on a molar basis) than is the DNA injected by phage particles; this result is apparently due to inactivating events suffered by transfecting DNA during its metabolism by competent cells. Earlier studies also demonstrated that, in some cases, the infectivity of transfecting DNA can be increased by ultraviolet (UV) irradiation of the competent cells prior to transfection, or by cotransfection of UV-irradiated heterologous DNAs; collectively, these phenomena have been termed transfection enhancement (TE). We propose here that some transfecting B. subtilis phage DNAs are attacked by a novel host DNA repair system, and that TE reflects inhibition of this by a competing substrate in UV-irradiated DNA. In support of this model, we show that UV-DNA cotransfection leads to a reduced rate of intracellular endonucleolytic breakdown of transfecting DNA. We also demonstrate that TE displays marked specificity of a kind frequently observed for repair enzymes. Thus, phages that contain hydroxymethyl uracil (HMU), but not thymine, in their genomes are susceptible to this process. In addition, we show that the photoproduct(s) in UV-irradiated DNA that produces TE by cotransfection is specific, and is not uracil, a pyrimidine dimer, thymine glycol, HMU, or a substrate for the E. coli thymine glycol DNA N-glycosylase. This photoproduct is derivable from thymine or HMU. The implications of these results are discussed.

Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway. II: Host constitutive expression, repair DNA synthesis, and in vitro activity.

In the Bacillus subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, sometimes followed by productive cell infection. Previous studies have shown that ultraviolet (UV)-irradiation of the competent host cells, or cotransfection of UV-irradiated heterologous DNA, can increase the efficiency of transfection in some cases; these latter two phenomena have been called transfection enhancement (TE). In an accompanying paper, we show that TE is apparently confined to the B. subtilis phages that contain hydroxymethyluracil (HMU) in their DNA, and that the photoproduct in UV-irradiated DNA that mediates TE is specific, and different than the pyrimidine dimer, thymine glycol, uracil, or HMU. We also show that TE is due to reduced intracellular endonucleolytic attack of transfecting DNA. Based on this DNA base and nucleolytic specificity, we hypothesized that TE reflects the incidental action of a host DNA repair system on transfecting HMU phage DNA. In continuing these studies, we show here that duplex infecting HMU phage DNA is apparently inactivated by this same putative repair system when phage protein synthesis is blocked. We find, too, that this inactivation of infecting HMU phage DNA can be inhibited by UV-irradiated DNA, and that this process has a similar DNA base specificity as for TE. The survival of infecting HMU phage DNA is dependent on host DNA polymerase activity. We can detect specific DNA synthesis consistent with formation of repair patches when inactivation of infecting HMU phage DNA is ongoing, but not when it is inhibited by the presence of UV DNA or by allowing phage gene expression. Each of these results is consistent with the hypothesis that TE reflects the action of a novel DNA repair pathway. We show that a candidate TE-associated enzymatic activity can be detected in cell free extracts of uninfected, but not HMU phage-infected, B. subtilis cells. Correspondingly, the extracts of phage-infected cells appear to contain a diffusible factor that acts as an inhibitor of this host enzyme.

Lack of dependence of 5-fluorodeoxyuridine-mediated radiosensitization on cytotoxicity.

It has been proposed that fluoropyrimidine-mediated cytotoxicity and radiosensitization are closely correlated. We have shown that HT29 human colon cancer cells transfected with the E. coli dUTPase gene are resistant to 5-fluorodeoxyuridine (FdUrd)-mediated cytotoxicity, presumably through more effective elimination of dUTP. We used these cells to assess the association between radiosensitization and cytotoxicity produced by FdUrd. The radiation sensitivities of the clones expressing elevated dUTPase activity (dutE clones) were similar to those of untransfected HT29 cells or HT29 cells which had been transfected with only the expression vector for the E. coli gene (con clones). We found that FdUrd produced similar increases in radiation sensitivity regardless of dUTPase activity. Levels of dUTPase in the dutE clones remained elevated during the entire period of FdUrd exposure, demonstrating that the lack of difference between dutE and Con clones was not a reflection of down-regulation of dUTPase activity by FdUrd. Flow cytometry showed that all clones progressed past the G1/S-phase boundary and into early S phase during FdUrd treatment. These data suggest that the mechanisms of FdUrd-mediated cytotoxicity and radiosensitization are not closely linked. These findings, combined with our previous investigations, are consistent with the hypothesis that radiosensitization occurs in cells which progress past the G1/S-phase boundary in the presence of FdUrd.

Low grade gliomas: preliminary analysis of failure patterns among patients treated using 3D conformal external beam irradiation.

PURPOSE: The pattern of failure of low grade gliomas following radiotherapy is less well known than that of the high grade gliomas. Stereotactic histologic studies have suggested that tumor cells extend beyond imaging abnormalities, and that large margins would be required for radiotherapy target volumes to encompass all of the neoplasm. Our experience using computerized tomography (CT)- and magnetic resonance (MR)-planned irradiation of low grade gliomas was reviewed to determine the pattern of tumor recurrence, in an effort to clinically define the minimum margin required. METHODS AND MATERIALS: Forty-six patients with low grade supratentorial gliomas were treated between April 1985 and November 1992 using three-dimensional (3D) conformal CT- or MR-planned external beam radiotherapy. Fields were designed to encompass a target volume created by adding a margin to the tumor in three dimensions. Generally, patients were treated using shrinking fields with an initial target (tumor plus a 1 to 3 cm margin) treated to a dose of 45 to 50.4 (median 50.4) Gy, and a boost (tumor plus a 0 to 2 cm margin) treated to a total of 54 to 59.4 (median 59.4) Gy. Median follow-up was 32.9 months. RESULTS: There have been 11 failures; all of these occurred within the radiographic abnormality (either T2 prolongation or CT hypodensity) visualized at the time of treatment planning (i.e., all failures were within the boost volume). Median time to failure was 53 months. Because all failures were local, there was no relationship between the amount by which the tumor volumes were expanded to create target volumes and the eventual outcome. CONCLUSION: Despite pathologic data suggesting that low grade glioma cells can be found outside the MR T2-signal abnormality in many cases, our results demonstrate that conformal external beam radiotherapy, in which the high dose volume is limited, does not result in increased marginal or out-of-field failures. Until control of tumor within the radiographically abnormal volume can be achieved, the need for large fields to treat prophylactically microscopic disease beyond the visualized tumor volume is questionable. The use of conformal fields might be associated with reduced toxicity, and thereby allow delivery of higher total doses to the central tumor.

Genome-wide search for loss of heterozygosity in transgenic mouse tumors reveals candidate tumor suppressor genes on chromosomes 9 and 16.

A genome-wide scan for loss of heterozygosity (LOH) in tumors provides a powerful route to the identification of genes involved in tumorigenesis. This approach has not previously been applied to transgenic mice, despite the considerable advantages they afford for genetic dissection. Here, we report a genome-wide LOH analysis of insulinomas and carcinoid tumors in transgenic mice expressing the simian virus 40 large tumor oncogene. Although the overall genome-wide rate of LOH was quite low, chromosomes 9 and 16 showed high rates of allelic loss. About one-third of tumors showed partial LOH, allowing localization of the likely tumor suppressor genes to intervals of approximately 11 centimorgans. The locus on chromosome 9, named Loh-1, lies in a region with synteny conservation to human chromosomes 3q, 6q12, 15q24, and 3p21, while the locus on chromosome 16, named Loh-2, lies in a region corresponding to human chromosomes 3q and 22q. Of particular note is the synteny conservation with human 3p21, which shows frequent loss in human cancers. These regions do not encode two tumor suppressors, pRB and p53, known to interact with large tumor oncoprotein, suggesting the presence of new genes whose loss of function contributes to multistage tumorigenesis.

Induction of resistance to fluorodeoxyuridine cytotoxicity and DNA damage in human tumor cells by expression of Escherichia coli deoxyuridinetriphosphatase.

Recent studies from our laboratory suggested that, in some human colorectal tumor cell lines, sensitivity to fluorodeoxyuridine may depend upon the extent of dUTP accumulation that occurs following drug treatment and that elevation of dUTPase activity might be the basis for some instances of resistance to fluoropyrimidines. To test this model, we expressed Escherichia coli dUTPase in an established human tumor cell line (HT29) and measured the effect of this manipulation on response to fluorodeoxyuridine. As predicted, HT29 derivatives containing dUTPase activity 4-5-fold higher than controls were protected from fluorodeoxyuridine-induced loss of clonogenicity and from formation of DNA double strand breaks. These data provide the first direct evidence that alteration in a component of the uracil misincorporation/misrepair pathway can confer resistance to fluoropyrimidines in human tumor cells.

Directed establishment of rat brain cell lines with the phenotypic characteristics of type 1 astrocytes.

Interest in obtaining cell lines for use in studies on the development and biochemistry of the central nervous system has motivated efforts to establish cells from primary brain cultures by the use of oncogene-transfer techniques. In previous reports, cell lines derived from astrocytes in this way have had immature or abnormal phenotypes. We have explored the possibility of specifically "targeting" expression of exogenous oncogenes to differentiated astrocytes by using the promoter of the gene encoding glial fibrillary acidic protein, which is expressed almost exclusively in such cells. We report here that cell lines displaying the phenotypic characteristics of type 1 astrocytes can be established reproducibly in this manner. Given the heterogeneity of primary cultures, the availability of clonal cell lines displaying characteristics of type 1 astrocytes should greatly facilitate our understanding of the biology of these cells.

Activities involved in base excision repair of bacteriophage T4 and lambda DNA in vivo.

The in vivo excision repair functions of Escherichia coli exonuclease III and 3-methyladenine DNA glycosylase I, and bacteriophage T4 pyrimidine dimer-DNA glycosylase were investigated. Following exposure of bacteriophage T4 or lambda to methyl methanesulfonate or ultraviolet irradiation, survival was determined by plating on E. coli have various genetic backgrounds. Although exonuclease III was shown to participate in base excision repair initiated by 3-methyladenine DNA glycosylase I, it had no detectable role in base excision repair initiated by the T4 pyrimidine dimer-DNA glycosylase. Despite its 3' apurinic/apyrimidinic endonuclease activity in vitro, T4 pyrimidine dimer-DNA glycosylase, even in large quantities, did not complement mutants defective in exonuclease III in the repair of apurinic sites generated by 3-methyladenine DNA glycosylase I in vivo.

Disseminated extraintestinal isosporiasis in a patient with acquired immune deficiency syndrome.

This report describes a 38-year-old male homosexual with acquired immune deficiency syndrome (AIDS) and a history of Isospora belli infestation treated with trimethoprim-sulfamethoxazole. At autopsy, intracellular and extracellular I. belli organisms were identified in the mucosa and lamina propria of small and large intestine as well as in mesenteric and tracheobronchial lymph nodes, where an associated granulomatous reaction was also present. Until now, Isosporiasis has been observed as intracellular parasites restricted to the columnar cells of the intestinal mucosa in humans hosts. This is, to the authors' knowledge, the first time that this protozoa has been observed invading beyond the intestinal wall. This patient's autopsy also demonstrated intestinal ulcerations proven microscopically to be caused by cytomegalovirus (CMV). In patients with AIDS and CMV enterocolitis, the authors have observed an unusual frequency of extraintestinal disseminated disease by microorganisms heretofore typically restricted to the intestinal mucosa (i.e., bacteremias resulting from Shigella species). The authors propose that, in AIDS patients, the profound immunodeficient state, possibly in conjunction with CMV-induced intestinal mucosal ulcerations, promotes access of other intestinal microorganisms, including I. belli, into lymphatic and vascular spaces, culminating in lymphohematogenous dissemination of their intestinal infestations.

Physical mapping and complete nucleotide sequence of the denV gene of bacteriophage T4.

Phage T4 deletion mutants that are folate analog resistant (far) and contain deletions in the region of the T4 genome near denV have been isolated previously. We showed that one of these mutants (T4farP12) expressed normal denV gene activity, whereas another mutant (T4farP13) was defective in the denV gene. The rII-distal (right) physical endpoints of these deletions defined the limits of the interval in which the rII-proximal (left) endpoint of the denV gene should be located. The deletion endpoints were identified by restriction and Southern hybridization analyses of phage derivatives containing deoxycytidine instead of hydroxymethyldeoxycytidine in their DNAs. The results of these analyses localized the rII-proximal (left) end of the denV gene to a region between 62.4 and 64.3 kilobases on the T4 physical map. denV+ phage resulted from marker rescue with two of five denV- alleles tested, using plasmids containing a 1.8-kilobase fragment from this region or a 179-base-pair terminal fragment derived from it. Sequencing of the 179-base-pair fragment from wild-type DNA showed a 130-base-pair open reading frame with its termination codon at the rII-proximal end. Confirmation that this open reading frame is part of the denV coding sequence was obtained by identifying a TAG amber codon in the homologous DNA derived from a denV amber mutant strain. This mutant strain rescued the denV+ allele from plasmids containing the wild-type sequence. An adjacent overlapping restriction fragment was also cloned, permitting determination of the remaining denV gene sequence. Based on these results, the 3' end of the coding region of the denV locus was mapped to kilobase position 64.07 on the T4 physical map, and the 5' end was mapped to position 64.48.

Measurement of repair patch size by quantitation of nucleotides excised during DNA repair in vivo.

Escherichia coli uvr- cells, prelabeled in their DNA, were infected with phage T4 denV+ or T4 denV- under conditions that preclude phage-mediated degradation of the bacterial chromosome. Measurement of the distribution of acid-soluble radioactivity between pyrimidine dimers and nondimer nucleotides in cell extracts yielded calculated estimates of the average size of excision repair tracts that are in good agreement with the size of repair patches determined by others using direct measurement of repair synthesis.

Pyrimidine dimer-DNA glycosylases: studies on bacteriophage T4-infected and on uninfected Escherichia coli.

Pyrimidine dimer (PD)-DNA glycosylase activity has been reported in both the M. luteus and phage T4 UV endonucleases. In the present studies the T4 PD-DNA glycosylase has been purified close to physical homogeneity using an assay that measures the release of free thymine from UV-irradiated poly ([H5] dT):poly (dA), after the photo-reversal of thymine-thymine dimers. The activity has also been demonstrated in vivo following infection of UV-irradiated E. coli uvr- cells with phage T4. Under these conditions the T4 PD-DNA glycosylase accounts quantitatively for all thymine-containing PD excised from [3H] labeled E. coli DNA. In vitro the T4 PD-DNA glycosylase has an associated AP endonuclease activity that incises UV-irradiated DNA 3 to the apyrimidinic sites created by the glycosylase. However, the glycosylase/AP endonuclease reaction mechanism in vitro does not appear to be a concerted one. In addition, a T4 phage with a temperature-sensitive mutation in the denV gene shows wild-type levels of survival at the permissive temperature, despite the fact that in vitro, extracts of E. coli infected with this mutant show no detectable phage-coded AP endonuclease at 28 degrees C. Thus the exact role of the T4 AP endonuclease in the incision of UV-irradiated DNA dimer in vivo is not clear. The ratio of excised non-containing nucleotides to dimer-containing nucleotides following infection of UV-irradiated E. coli with phage T4 denV+ yields a calculated average repair patch size of approximately 7 nucleotides. In contrast, the calculated average patch size in uninfected E. coli is approximately 70 nucleotides. Thus the extent of excision/resynthesis of UV-irradiated DNA may be determined by the specific mode of incision of the DNA at PD. When uninfected E. coli (uvr+) is exposed to UV radiation, a fraction of the excised thymine-containing PD contain photolabile thymine, suggesting the presence of PD-DNA glycosylase in E. coli. The role of this putative activity in the metabolism of UV-irradiated DNA is under investigation.