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PubMed2005

Defining the roles of aromatase inhibitors in the adjuvant treatment of early-stage breast cancer.

The widespread use of tamoxifen has led to significant improvements in survival for postmenopausal women with early-stage hormone receptor-positive breast cancer; however, approximately 30% of patients die despite receiving tamoxifen as adjuvant treatment. In addition, concerns exist regarding tamoxifen-associated side effects, including endometrial cancer and thromboembolic disease. The development of the third-generation aromatase inhibitors (AIs; anastrozole, exemestane, and letrozole) therefore represented a welcome potential alternative to tamoxifen. Several clinical trials have demonstrated the superiority of AIs over tamoxifen in the adjuvant treatment of postmenopausal women with hormone-sensitive breast cancer, but these trials differ in their design and in the characteristics of their patient populations. This review discusses the different designs of the primary adjuvant, switching, extended adjuvant, and sequencing trials that are investigating the use of AIs in the adjuvant treatment of breast cancer and provides direction regarding how the data from these trials could be used to guide treatment choice. This review also demonstrates why one should not extrapolate results from clinical trials to clinical situations that differ from the clinical trial or from clinical trials investigating a particular AI to clinical situations involving another AI.

Anastrozole↗
PubMed1989

Reversion of trpA nonsense mutations by deletion of the chain-termination codons.

This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of Escherichia coli. This mechanism, deletion of the nonsense codon, was discovered in the course of selecting for missense revertants of trpA(UGA211) and for catalytically active tryptophan synthetase alpha chain revertants of trpA(UAA234) and trpA(UAG234). Each type of revertant trpA was cloned and its DNA sequence determined. trpA(UGA211) gave rise to two previously unidentified types of missense revertant. The first type was expected, namely trpA(CGA211), the result of a base substitution event. The other type, representing approximately 1% of the missense revertants, was unexpected on the basis of single base substitutions and an understanding of which amino acids are functional at alpha chain position 211. It was found to be the result of a 21 base-pair deletion of a region containing codon 211. The tryptophan-independent revertants of both position 234 nonsense mutants occurred at a frequency of approximately 2 per 10(9) viable cells. They were identical in that they both resulted from a 3 base-pair deletion, namely deletion of the chain-terminating codon at position 234. One of them, however, also displayed an A instead of the normal G in the third position of codon 235. The revertants were characterized according to growth in different media and tryptophan synthetase assays performed on crude extracts. These types of mutants should prove interesting and important for the elucidation of alpha chain structure-function relationships, for insight into the assembly and interaction of subunits in this model multienzyme complex, and for the study of mechanisms by which deletions can be generated.

Base Sequence↗
PubMed1993

Repression of hybrid dysgenesis in Drosophila melanogaster by individual naturally occurring P elements.

Individual P elements that were genetically isolated from wild-type strains were tested for their abilities to repress two aspects of hybrid dysgenesis: gonadal dysgenesis and mutability of a double-P element-insertion allele of the singed locus (snw). These elements were also characterized by Southern blotting, polymerase chain reaction amplification and DNA sequencing. Three of the elements were 1.1-kb KP elements, one was a 1.2-kb element called D50, and one was a 0.5-kb element called SP. These three types of elements could encode polypeptides of 207, 204, and 14 amino acids, respectively. Gonadal dysgenesis was repressed by two of the KP elements (denoted KP(1) and KP(6)) and by SP, but not by the third KP element (KP(D)), nor by D50. Repression of gonadal dysgenesis was mediated by a maternal effect, or by a combination of zygotic and maternal effects generated by the P elements themselves. The mutability of snw was repressed by the KP(1) and KP(6) elements, by D50 and by SP, but not by KP(D); however, the SP element repressed snw mutability only when the transposase came from complete P elements and the D50 element repressed it only when the transposase came from the modified P element known as delta 2-3. In all cases, repression of snw mutability appeared to be mediated by a zygotic effect of the isolated P element. Each of the isolated elements was also tested for its ability to suppress the phenotype of a P-insertion mutation of the vestigial locus (vg21-3). D50 was a moderate suppressor whereas SP and the three KP elements had little or no effect. These results indicate that each isolated P element had its own profile of repression and suppression abilities. It is suggested that these abilities may be mediated by P-encoded polypeptides or by antisense P RNAs initiated from external genomic promoters.

Alleles↗
PubMed2005

Phenotypic and DNA sequence data comparisons reveal three discrete species in the Ceratocystis polonica species complex.

Ceratocystis polonica and C. laricicola are two morphologically similar species that occur on conifers and reside in the Ceratocystis coerulescens species complex. They, however, represent two ecologically distinct entities. C. polonica causes blue stain on Norway spruce (Picea abies) and other spruce species (Picea spp.) in Eurasia and is associated with the bark beetles Ips typographus, I. typographus japonicus, I. amitinus and I. duplicatus. In contrast, C. laricicola lives in a symbiotic relationship with the bark beetles Ips cembrae and I. subelongatus that infest various larch species (Larix spp.). The objective of this study was to consider the phylogenetic relationships of C. polonica and C. laricicola and more specifically to determine the identity of Japanese isolates from both spruce and larch, based on sequences derived from the ITS regions of the rRNA operon, the beta-tubulin gene and the HMG box of the MAT-2 gene. Isolates were also compared based on morphology and cultural characteristics. Comparisons of anamorph and teleomorph structures confirmed that C. polonica and C. laricicola are indistinguishable based on morphology. Both species had an optimal growth temperature of 25 degrees C. However, at temperatures between 31-33 degrees, C. polonica isolates grew slowly or not at all, while C. laricicola isolates grew more actively at these temperatures. Thus, a growth test at 32 degrees can differentiate these species. Phylograms generated using parsimony for the three gene regions were strongly congruent. These showed three distinct clades supported by high bootstrap values. Two of the clades clearly separate C. laricicola from Europe and C. polonica, supporting the view that they represent two discrete taxa. A third clade included isolates obtained from galleries of Ips subelongatus on Larix kaempferi in Japan. This fungus clearly represents a discrete taxon that is closely related to, but distinct from C. laricicola, which is described here as C. fujiensis sp. nov.

Animals↗
PubMed1975

Translocation of a plasmid DNA sequence which mediates ampicillin resistance: molecular nature and specificity of insertion.

A series of recombinant plasmids was generated in Escherichia coli in which the TEM beta-lactamase translocon (TnA) was inserted into the small plasmid RSF1010. RSF1010 is a 5.5 X 10(6) dalton nonconjugative plasmid which confers resistance to streptomycin and sulfonamide. The recombinant plasmids can be classified into three clearly defined phenotypic groups. Group I is ampicillin-, streptomycin- and sulfonamide-resistant. Group II is ampicillin- and sulfonamide-resistant but has lost streptomycin resistance. Group III is ampicillin-resistant but is sensitive to sulfonamide and shows a simultaneous 30-fold reduction in the minimal inhibitory concentration of streptomycin. It was possible to map the site of insertion of TnA within RSF1010 by electron microscope studies of DNA heteroduplexes formed between RSF1010 and recombinant plasmids. Insertions of TnA occur at, at least, 12 distinct sites in a region corresponding to one-third of the RSF1010 DNA molecule. Those insertions giving rise to particular phenotypes are clustered. Insertions of TnA-like insertion sequences (IS) appear to give rise to strongly polar mutations.

Ampicillin↗
PubMed1992

Molecular analysis of Saccharomyces cerevisiae chromosome I. On the number of genes and the identification of essential genes using temperature-sensitive-lethal mutations.

Previous analyses of Saccharomyces cerevisiae chromosome I have suggested that the majority (greater than 75%) of single-copy essential genes on this chromosome are difficult or impossible to identify using temperature-sensitive (Ts-) lethal mutations. To investigate whether this situation reflects intrinsic difficulties in generating temperature-sensitive proteins or constraints on mutagenesis in yeast, we subjected three cloned essential genes from chromosome I to mutagenesis in an Escherichia coli mutator strain and screened for Ts- lethal mutations in yeast using the "plasmid-shuffle" technique. We failed to obtain Ts- lethal mutations in two of the genes (FUN12 and FUN20), while the third gene yielded such mutations, but only at a low frequency. DNA sequence analysis of these mutant alleles and of the corresponding wild-type region revealed that each mutation was a single substitution not in the previously identified gene FUN19, but in the adjacent, newly identified essential gene FUN53. FUN19 itself proved to be non-essential. These results suggest that many essential proteins encoded by genes on chromosome I cannot be rendered thermolabile by single mutations. However, the results obtained with FUN53 suggest that there may also be significant constraints on mutagenesis in yeast. The 5046 base-pair interval sequenced contains the complete FUN19, FUN53 and FUN20 coding regions, as well as a portion of the adjacent non-essential FUN21 coding region. In all, 68 to 75% of this interval is open reading frame. None of the four predicted products shows significant homologies to known proteins in the available databases.

Amino Acid Sequence↗
PubMed1987

Comparison of the reovirus serotype 1, 2, and 3 S3 genome segments encoding the nonstructural protein sigma NS.

The sequences of the S3 genome segments of reovirus serotype 1 and 2 prototype strains are presented and these sequences are compared with the sequence of the serotype 3 S3 genome segment. The S3 genome segment encodes the nonstructural protein sigma NS which possesses affinity for ssRNA and appears to function in reovirus morphogenesis. The three S3 genome segments are closely related: all are 1198 nucleotides long and possess a single long open reading frame 366 codons long. They exhibit a serotype 1:3 relatedness pattern: there are only 13% mismatches between the S3 genome segments of serotypes 1 and 3, but 27 and 26% mismatches, respectively, between those of serotype 1 and 2 and serotype 3 and 2. The amino acid mismatches for the three sigma NS proteins are much lower (2.7, 13.9, and 13.7%, respectively), because the majority of nucleotide mismatches are in third base codon positions. The three sigma NS proteins possess a conserved secondary structure that is rich in alpha-helix content; in fact, the predicted alpha-helix content of these nonstructural proteins (about 50%) is much higher than that of the three other sigma size class proteins (about 20%), all of which are structural proteins. We also sequenced the S3 genome segment of a ts mutant of serotype 3 generated by treatment with nitrous acid and found a single nucleotide change that specifies an amino acid change that introduces a five-residue-long beta-sheet prone configuration into a long (80 amino acids) highly conserved alpha-helix in the C-terminal half of sigma NS. This change could account for this mutant's ts character. Finally, the three sigma NS proteins have diverged in only about 10% of positions, whereas the three sigma 1 proteins have diverged in about 70%. The rapid evolutionary divergence of the latter may be a result of several factors, including (i) the fact that sigma 1, but not sigma NS, is under immunologic selective pressure; (ii) the fact that the functions of sigma 1 (antigenicity and cell attachment) probably reside in two rather small domains that are not restricted spatially with respect to each other; and (iii) the fact that the functions of sigma NS, namely RNA binding and protein binding (during morphogenesis), require a highly specific overall protein configuration that may permit little variation.

Amino Acid Sequence↗
PubMed1979

Sequence analysis of adenovirus DNA: complete nucleotide sequence of the spliced 5' noncoding region of adenovirus 2 hexon messenger RNA.

The complete nucleotide sequence of the 5' noncoding region of the adenovirus 2 hexon messenger RNA has been established by sequence analysis of reverse transcripts. Such transcripts were generated by extension of specific single-stranded DNA primers with reverse transcriptase after hybridization to purified hexon mRNA. The total length of the 5' noncoding region was determined to be 240 nucleotides, of which the spliced tripartite leader sequence contributes 202 nucleotides including the terminal m7G. The sizes of the different segments of the tripartite leader were estimated by comparing the established mRNA sequence with the genomic sequences for the first and third leader segments, and were found to be 42 nucleotides for the first segment, 71 nucleotides for the second and 89 nucleotides for the third. The estimates are ambiguous, however, due to the presence of tandemly repeated sequences at both ends of the intervening sequence between the third leader segment and the body of the hexon mRNA. The sequence of the leader allows the formation of hydrogen-bonded interactions with the 3' end of 18S ribosomal RNA near the capped 5' end and also close to the initiator AUG.

Adenoviruses, Human↗
PubMed2005

Functional analysis of fengycin synthetase FenD.

Fengycin is a cyclic lipopeptidic antibiotic produced nonribosomally by Bacillus subtilis. A fengycin synthetase mutant of B. subtilis F29-3 was generated with Tn917lux, which contains a transposon inserted in a 7716-bp gene, fenD. The mutation can be genetically complemented by transforming a plasmid carrying a wild-type fenD, confirming the participation of the gene in fengycin synthesis. Sequencing and biochemical analysis reveal that this gene encodes an enzyme that includes two amino acid-activating modules, FenD1 and FenD2, which activate l-Tyr and l-Thr, the third and the fourth amino acids in fengycin, respectively.

Amino Acid Sequence↗
PubMed2001

Chloroplasts have a novel Cpn10 in addition to Cpn20 as co-chaperonins in Arabidopsis thaliana.

Previously, we characterized a mitochondrial co-chaperonin (Cpn10) and a chloroplast co-chaperonin (Cpn20) from Arabidopsis thaliana (Koumoto, Y., Tsugeki, R., Shimada, T., Mori, H., Kondo, M., Hara-Nishimura, I., and Nishimura, M. (1996) Plant J. 10, 1119-1125; Koumoto, Y., Shimada, T., Kondo, M., Takao, T., Shimonishi, Y., Hara-Nishimura, I., and Nishimura, M. (1999) Plant J. 17, 467-477). Here, we report a third co-chaperonin. The cDNA was 603 base pairs long, encoding a protein of 139 amino acids. From a sequence analysis, the protein was predicted to have one Cpn10 domain with an amino-terminal extension that might work as a chloroplast transit peptide. This novel Cpn10 was confirmed to be localized in chloroplasts, and we refer to it as chloroplast Cpn10 (chl-Cpn10). The phylogenic tree that was generated with amino acid sequences of other co-chaperonins indicates that chl-Cpn10 is highly divergent from the others. In the GroEL-assisted protein folding assay, about 30% of the substrates were refolded with chl-Cpn10, indicating that chl-Cpn10 works as a co-chaperonin. A Northern blot analysis revealed that mRNA for chl-Cpn10 is accumulated in the leaves and stems, but not in the roots. In germinating cotyledons, the accumulation of chl-Cpn10 was similar to that of chloroplastic proteins and accelerated by light. It was proposed that two kinds of co-chaperonins, Cpn20 and chl-Cpn10, work independently in the chloroplast.

Amino Acid Sequence↗
PubMed2002

The first inner loop of endothelin receptor type B is necessary for specific coupling to Galpha 13.

Endothelin (EDN) receptor type B (EDNRB) activates serum response factor (SRF) via G(q/11) and G(12/13) G proteins. In this study, we investigated the involvement of intracellular loop sequences of EDNRB in coupling to these G proteins. EDNRB mutants were generated and tested for their abilities to activate SRF in NIH3T3 cells and in the mouse embryonic fibroblast cell line (F(q/11)) lacking both Galpha(q) and Galpha(11). EDNRB can activate SRF in NIH3T3 cells via G(q/11), although it can only activate SRF through G(12/13) in F(q/11) cells. Mutants with mutations in the second and third inner loops of EDNRB functioned in the same manner in both cell lines, either able or unable to activate SRF. This finding suggests that the second and third inner loops of EDNRB either participate or not in coupling to both G(q/11) and G(12/13) but are not specific for either one. However, in the first inner loop, a substitution of three Ala residues for Met(128)-Arg(129)-Asn(130) abolished the ability to activate SRF only in F(q/11) cells, suggesting that this mutation might specifically disrupt the coupling to G(12/13) rather than to G(q/11). Further characterization of this first inner loop mutant revealed that exogenous expression of Galpha(12) or Galpha(q) could restore SRF activation, whereas the expression of Galpha(13) did not. Therefore, we conclude that although the three intracellular loops of EDNRB may be involved in coupling to G proteins, residues Met(128)-Arg(129)-Asn(130) in the first intracellular loop are specifically required for activation of Galpha(13).

3T3 Cells↗
PubMed2003

EnteriX 2003: Visualization tools for genome alignments of Enterobacteriaceae.

We describe EnteriX, a suite of three web-based visualization tools for graphically portraying alignment information from comparisons among several fixed and user-supplied sequences from related enterobacterial species, anchored on a reference genome (http://bio.cse.psu.edu/). The first visualization, Enteric, displays stacked pairwise alignments between a reference genome and each of the related bacteria, represented schematically as PIPs (Percent Identity Plots). Encoded in the views are large-scale genomic rearrangement events and functional landmarks. The second visualization, Menteric, computes and displays 1 Kb views of nucleotide-level multiple alignments of the sequences, together with annotations of genes, regulatory sites and conserved regions. The third, a Java-based tool named Maj, displays alignment information in two formats, corresponding roughly to the Enteric and Menteric views, and adds zoom-in capabilities. The uses of such tools are diverse, from examining the multiple sequence alignment to infer conserved sites with potential regulatory roles, to scrutinizing the commonalities and differences between the genomes for pathogenicity or phylogenetic studies. The EnteriX suite currently includes >15 enterobacterial genomes, generates views centered on four different anchor genomes and provides support for including user sequences in the alignments.

Computer Graphics↗
PubMed1996

Genome plasticity in Neisseria gonorrhoeae.

The pathogenic Neisseria have exploited the processes of horizontal DNA transfer and genetic recombination as mechanisms for the generation of extensive protein variation and modulation of gene expression. Localized recombinations have been well documented in members of multigene families as have alterations in short repetitive sequences. Here we report an analysis of the chromosomal structure of a defined lineage of Neisseria gonorrhoeae strain MSl1 pilin variants. This study reveals the occurrence of large rearrangements, including the amplification of a 26 kb region and an inversion involving more than a third of the chromosome. Additionally, a restriction site polymorphism that correlates with pilin expression has been observed. These findings highlight the flexibility of the gonococcal genome.

Bacterial Outer Membrane Proteins↗
PubMed2006

Primary mediastinal large B-cell lymphoma: a single-center study of clinicopathologic characteristics.

Primary mediastinal large B-cell lymphoma (PMLBCL) is a subset of LBCL with unique clinicopathologic features. Some studies have raised the question of differences in biological features and clinical course among patients from different parts of the world. We conducted a retrospective clinicopathologic analysis of 24 patients with PMLBCL from a single center in Croatia. We also conducted the first investigation of the frequency of lymphotropic viruses human herpesvirus 6 (HHV-6) and HHV-8 in lymphoid lesions of this disease. The clinical characteristics of the patients were as expected, with high International Prognostic Index scores, elevated serum lactate dehydrogenase (LDH) levels, and bulky disease being adverse prognostic factors. Only 6 patients (25%) showed CD30 expression, and Bcl-6 protein expression was, in our series, prognostically favorable (P = .0401). One patient's tumor had detectable HHV-6 genome sequence, but no HHV-8 sequences were detected in any tumors. Two thirds of the patients received CHOP chemotherapy (cyclophosphamide, hydroxydaunomycin, vincristine, and prednisone) with a relatively low complete remission rate (43.8%; median follow-up, 33.8 months). This study confirmed the moderate preponderance among PMLBCL patients of young females with B symptoms and elevated LDH levels. The CHOP regimen proved effective as first-line therapy only in patients with limited disease. Therefore, other third-generation chemotherapy protocols may be considered for treatment, especially in patients with bulky and advanced disease.

Adult↗
PubMed1993

Nucleotide sequence analysis of the VL and VH domains of five human IgM directed to lamin B. Evidence for an antigen-driven process in the generation of human autoantibodies to lamin B.

OBJECTIVE: To gain insight into the genetic origin of human antilamin autoantibodies, we determined the nucleotide sequence of the light and heavy chain variable region (VL and VH) domains of 5 IgM antibodies directed to lamin B. These antibodies represent a distinct subset of antinuclear antibodies, and their presence is associated with a particular lupus-like syndrome. METHODS: We derived and cloned lymphoblastoid cell lines from peripheral blood B cells of 3 patients, selected anti-lamin B-producing subclones, and sequenced the messenger RNA coding for Ig heavy and light chains. RESULTS: We isolated 2 subclones (1 IgM kappa, 1 IgM lambda) from one patient (FUR) and 2 subclones (both IgM lambda) from another (HER). In contrast, all 8 lines derived from B cells isolated from the third patient (BEN) synthesized identical anti-lamin B IgM kappa antibodies: All VL and VH domains from these 5 IgM were encoded by different VL or VH genes. DH regions were all different, and there was no restriction in the use of JL or JH segments. Analysis of the nucleotide sequence of the VL domains allowed the identification of the putative germinal gene in 3 instances (V kappa IV, Humkv325, and V lambda III.1); the overall ratios of replacement:silent mutations (R:S) were 6.5 and 1.2 in the complementarity-determining regions (CDRs) and framework regions (FRs), respectively. The 2 other lambda sequences belonged to the V lambda III family. With regard to VH domains, 3 of 5 derived from previously identified germline genes (VHIV 4.19, VHIV 4.22, and VHIII 9.1); the overall R:S ratio for these genes was 8 and 1.5 in CDRs and FRs, respectively. CONCLUSION: Taken together, these data provide evidence that the repertoire of human antilamin autoantibodies is not restricted and that the antigen (or another kind of selective pressure) plays a role in the generation of autoantibodies to lamin B. This hypothesis is in accordance with the reactivity of these antibodies to discrete epitopes of lamin B.

Antigens↗
PubMed1995

Genetic self-management in a captive colony of mandrills (Mandrillus sphinx) as revealed by DNA minisatellite fingerprints.

DNA fingerprints using three oligonucleotide probes, a (GTG)5 short tandem repeat sequence, and 2 corresponding to the core sequences of minisatellites 33.15 and 33.6, have been used to generate DNA fingerprints of a captive colony of mandrills (Mandrillus sphinx) housed at the Centre International de Recherches Médicales de Franceville (CIRMF), in order to monitor paternity. A colony of 6 males and 7 females was established in a 6 ha. rain-forested enclosure in 1983. Band sharing scores indicated that none of the founders were related. By April 1994 the colony had increased to 84 individuals. Forty-nine of the 77 births were first-generation infants, a further 27 were second-generation offspring, and one was a third-generation mandrill. Five animals died during the study, and one was removed for husbandry reasons. Overall band sharing between individuals increased markedly and the power to discriminate between first- and second-generation infants was reduced; the probability of two individuals sharing the same pattern increased 1000-fold from 10(-6) to 10(-3). The dominant male in the group effectively guards access to all receptive females during the annual breeding season. Paternity analysis confirmed that the dominant male is the father of the great majority of offspring born during this term of dominance. Daughters born before 1991 to either of the two dominant males reached sexual maturity during this tenure. Incestual inbreeding occurred with both males, but only 25% of all possible cases resulted in the birth of a viable infant; one male fathered infants on 3 of 13 possible occasions and a second male on 2 of 7 occasions. Female avoidance strategies and shifts in male dominance in this closed mandrill colony appeared to be timed in a way that reduces incest.

Animals↗
PubMed1981

5'-Terminal nucleotide sequence of Semliki forest virus 18S defective interfering RNA is heterogeneous and different from the genomic 42S RNA.

An 18S defective interfering (DI) RNA population was isolated from the cytoplasm of baby hamster kidney (BHK-21) cells infected with Semliki Forest virus from the 10th undiluted passage. The RNA was approximately 2000 nucleotides long and contained a 5'-terminal cap with the structure 7mGpppAp and a poly(A) tract. The DI RNA contained large TI oligonucleotides derived from both the 42S RNA-specific region and the 3' one-third of the genome common to 42S and 26S RNA. Several of the large oligonucleotides were present in nonequimolar ratios, suggesting that the RNA population is heterogeneous. As this population is approximately uniform in size, this suggests that the DI RNAs may be generated by internal deletions involving different regions of the genome. The 5'-terminal cap-containing RNase T1 oligonucleotide was isolated by two-dimensional gel electrophoresis from uniformly 32P-labeled RNA and shown to be heterogeneous. Five T1 caps with the structure 7mGpppA-U(A-U)nC-A-U-G(n = 4-8) were identified. The two major T1 caps (n = 4 and 6) comprised about 75% of the total yield of T1 caps. The T1 caps were different from the genomic 42S RNA T1 cap (7mGpppA-U-G), suggesting that the extreme 5' end of the genome is not conserved in this defective interfering RNA.

Base Sequence↗
PubMed2000

In vivo transgenic mutation assays.

Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage-based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3 x 10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two-thirds the MTD, and one-third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control.

Animals↗