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PubMed2004

Sympatric speciation by sexual selection alone is unlikely.

According to Darwin, sympatric speciation is driven by disruptive, frequency-dependent natural selection caused by competition for diverse resources. Recently, several authors have argued that disruptive sexual selection can also cause sympatric speciation. Here, we use hypergeometric phenotypic and individual-based genotypic models to explore sympatric speciation by sexual selection under a broad range of conditions. If variabilities of preference and display traits are each caused by more than one or two polymorphic loci, sympatric speciation requires rather strong sexual selection when females exert preferences for extreme male phenotypes. Under this kind of mate choice, speciation can occur only if initial distributions of preference and display are close to symmetric. Otherwise, the population rapidly loses variability. Thus, unless allele replacements at very few loci are enough for reproductive isolation, female preferences for extreme male displays are unlikely to drive sympatric speciation. By contrast, similarity-based female preferences that do not cause sexual selection are less destabilizing to the maintenance of genetic variability and may result in sympatric speciation across a broader range of initial conditions. Certain groups of African cichlids have served as the exclusive motivation for the hypothesis of sympatric speciation by sexual selection. Mate choice in these fishes appears to be driven by female preferences for extreme male phenotypes rather than similarity-based preferences, and the evolution of premating reproductive isolation commonly involves at least several genes. Therefore, differences in female preferences and male display in cichlids and other species of sympatric origin are more likely to have evolved as isolating mechanisms under disruptive natural selection.

Animals↗
PubMed2005

[DNA/MVA combined immunization: antibody response to Plasmodium falciparum merozoite surface protein 1 in mice].

OBJECTIVE: To explore the effect of DNA/MVA combined immunization in enhancing antibody response to MSP1. METHODS: DNA vaccine and recombined MVA were constructed based on synthesized MSP1 gene (3D7). BALB/c mice were primed with DNA solely or together with GM-CSF expressing plasmid and then boosted with rMVA/ 190. Serum IgG and subtype IgG1 and IgG2a were assayed by ELISA. All mice were challenged with allelic replaced Plasmodium berghei. RESULTS: Antibodies to MSP1-190 were detected after DNA immunization with an end-point dilution titer of 1:2500. When GM-CSF plasmid was added, the antibody end-point dilution titer reached 1:11150, with an increase of 53 and 10 times respectively after MVA boosting. Among them anti-19000 antibodies were prominent, 1/4-1/3 of total IgG in serum. However, when the mice were challenged with Pb-PfM19 no prolonged survival was observed (P>0.05). CONCLUSION: High titer antibodies can be elicited in mice by using codon optimized MSPI gene and DNA/MVA combined immunization. The specificity and protection of these antibodies is being further investigated.

Animals↗
PubMedDate not supplied

[Genetic factors predisposing to the development of papillary thyroid cancer].

According to classic theory of neogenesis, cancer arises from well-differentiated cell that in response to variety of factors de-differentiates, becomes able to proliferate without control and/or loses its ability to undergo apoptosis. According to another theory, cancers (at least cancers of some organs) originate from stem cells, which "by definition" are poorly differentiated and able to proliferate indefinitely. Therefore a lower number of abnormal events is necessary for these cells to escape proliferation-controlling mechanisms. With regard to papillary thyroid cancers it is still thought that it arises from well-differentiated thyreocyte. One of the characteristic features of cancer cell is chromosomal instability. Lowest number of such abnormalities is observed in well-differentiated thyroid cancers (including papillary cancer), intermediate - in poorly-differentiated cancers, while highest - in anaplastic cancers. Microarray analysis shows that despite of clinical heterogeneity, gene expression profiles of papillary cancers are very similar. Genetic anomalies predisposing to the development of papillary cancer most commonly regard proteins that possess kinase activity. Kinases phosphorylate other proteins, and play an extremely important role in signal transduction from outside the cell as well as inside the cell. Constitutive activation of some kinases may lead to the excessive and/or permanent activation of some transduction pathways specific for mitogens or growth factors. This results in excessive proliferation. The best known protein of such type which function is altered in papillary thyroid cancers is RET - a membrane-located growth factor-receptor with kinase activity. RET gene undergoes different rearrangements in this type of cancer. There are approximately 10 RET rearrangements known, with RET/PTC3 and RET/PTC1 being most common. In this anomaly kinase domain-encoding 3' end of RET gene is aberrantly bound to 5' end of another gene. Fusion protein synthesized on such hybrid template is not present in the cell membrane but in the cytoplasm, where it permanently activates transduction pathway specific for RET. NTRK1 gene encoding a member of family of neuronal growth factor receptors containing thyrosine kinase domain is also rearranged in papillary cancers. However, genes fused to its kinase domain-encoding sequence are different from the ones fused to RET. MET, a gene encoding another membrane protein with thyrosine kinase activity, which acts as a growth factor-receptor, is overexpressed in 70%-90% of papillary thyroid cancers. BRAF gene encoding another yet kinase transducing signals from RAS and RAF to the cell is mutated at position 1796 (T/A, amino acid substitution V599E) in 38-69% of papillary cancers. The presence of this activatory mutation is associated with higher degree of clinical advancement of the disease. In addition, in majority of papillary cancers tested, mutations of the genes encoding nuclear triiodothyronine receptors were found. Transgenic mice with both TRB allele replaced with dominant-negative TRB mutants develop aggressive thyroid cancers. Progression from papillary to anaplastic cancer is most possibly caused by the occurrence of additional anomalies within P53, RAS, NM23,b-catenin gene and other genes.

Adenocarcinoma, Papillary↗
PubMed2006

Neurotoxicity from innate immune response is greatest with targeted replacement of E4 allele of apolipoprotein E gene and is mediated by microglial p38MAPK.

Inheritance of APOE alleles is associated with varying clinical outcomes in several neurodegenerative diseases that are associated with innate immune response in brain. We tested the hypothesis that inheritance of different APOE alleles would significantly modulate neurotoxicity arising from glial innate immune response. We first used dissociated cultures of wild-type (wt) murine neurons and glia derived from mice with targeted replacement (TR) of the epsilon2, epsilon3, or, epsilon4 APOE allele. Our results showed that the vast majority of bystander damage to wt neurons derived from microglia was greatest with TR APOE4 glia, intermediate from TR APOE3 glia, and least from TR APOE2 glia and preceded detectable NO secretion. Microglial p38MAPK-dependent cytokine secretion followed a similar pattern of TR APOE dependence. In hippocampal slice cultures, innate immune activation had a similar pattern of TR APOE-dependence and produced postsynaptic neuronal damage in TR APOE4 and TR APOE3 but not TR APOE2 cultures that was p38MAPK dependent. These findings suggest a new mechanism by which inheritance of different APOE alleles may influence the outcome of neurodegenerative diseases associated with microglial innate immune response.

Alleles↗
PubMed2002

Functional versus structural matching: can the CTLp test be replaced by HLA allele typing?

Human leukocyte antigen (HLA) incompatibilities are the most important immunological barriers to bone marrow transplant success when using unrelated donors. Until recently, standards for donor selection included serological methods for HLA class I antigens and DNA-based typing for HLA class II alleles. In our center cytotoxic T-lymphocyte precursor (CTLp) assays have been an integrated part of the search selection procedure as well. More recently, DNA-based typing for HLA class I became available. This allowed us to determine the correlation of CTLp frequencies directed against incompatibilities at the HLA-A, -B, and -C locus in 211 donor-recipient pairs. HLA class I incompatibilities are significantly (p < 0.001) associated with high CTLp frequencies. Exceptions did occur, high CTLp frequencies are seen in 14% of the HLA-A, -B, and -C allele matched pairs, whereas in 7% of the pairs mismatched for HLA-A or -B a low CTLp frequency occurred. The successful outcome of transplants performed in the latter cases suggest that the CTLp test can be used as a tool to detect permissible mismatches when no fully matched donor is available. The influence of HLA-C mismatches on the CTLp outcome was less clear. Although in the majority of mismatched pairs (64%) the CTLp frequency was high, in 36% of the pairs the CTLp frequency was low. Analysis of HLA amino acid sequences was performed on the HLA-C allele mismatched group. An amino acid difference was always found at five polymorphic positions 97, 99, 113, 114, and 116 situated at the peptide binding groove in the high CTLp frequency group, whereas in the low CTLp frequency group this was observed in only 9 of 17 combinations (p < 0.001). However, this is mainly due to Cw*0303-0304 mismatches. In conclusion, although there is a highly significant correlation between the outcome of the CTLp frequency test and HLA allele class I typing, exceptions occur. It is unclear whether they are all clinically relevant but they certainly provide additional insight in allograft recognition.

Adult↗
PubMed1985

Direct and indirect gene replacements in Aspergillus nidulans.

We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC- strain to trpC+. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC- allele in a minority of the strains. An A. nidulans trpC+ gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC- argB+ strain to trpC+. Approximately 30% of the transformants were simultaneously argB-. The null argB allele had replaced the wild-type allele in a majority of these strains. The A. nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC- strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC- progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae.

Aspergillus nidulans↗
PubMed1989

Cloning of the flagellin gene from Bacillus subtilis and complementation studies of an in vitro-derived deletion mutation.

The flagellin promoter and structural gene from Bacillus subtilis I168 was cloned and sequenced. The amino-terminal protein sequence deduced from the coding sequence of the cloned gene was identical to that of the amino terminus of purified flagellin, indicating that the export of this protein is not directed by a posttranslationally processed N-terminal signal peptide. A sequence that was homologous to that of a consensus sigma 28 RNA polymerase recognition site lay upstream of the proposed translational start site. Amplification of this promoter region on a multicopy plasmid resulted in the formation of long, filamentous cells that accumulated flagellin intracellularly. The chromosomal locus containing the wild-type flagellin allele was replaced with a defective allele of the gene (delta hag-633) that contained a 633-base-pair deletion. Transport analysis of various flagellin gene mutations expressed in the hag deletion strain suggest that the extreme C-terminal portion of flagellin is functionally involved in export of the protein.

Amino Acid Sequence↗
PubMed2005

Hippocampal spatial memory impairments caused by the familial Alzheimer's disease-linked presenilin 1 M146V mutation.

Mutations in presenilins (PS) 1 and 2 are the major cause of familial Alzheimer's disease. Conditional inactivation of PS1 in the mouse postnatal forebrain leads to mild deficits in spatial learning and memory, whereas inactivation of both PS1 and PS2 results in severe memory and synaptic plasticity impairments, followed by progressive and substantial neurodegeneration. Here we investigate the effect of a familial Alzheimer's disease-linked PS1 missense mutation using knock-in (KI) mice, in which the wild-type PS1 allele is replaced with the M146V mutant allele. In the Morris water maze task, PS1 KI mice at 3 months of age exhibit reduced quadrant occupancy and platform crossing in the probe trial after 6 days of training, though their performance was normal in the probe trial after 12 days of training. By the age of 9 months, even after 12 days of training, PS1 homozygous KI mice still exhibit reduced platform crossing in the post-training probe trial. ELISA analysis revealed a selective increase in cortical levels of beta-amyloid 42 in PS1 KI mice, whereas production of beta-amyloid 40 was normal. Histological and quantitative real-time RT-PCR analyses showed normal gross hippocampal morphology and unaltered expression of three genes involved in inflammatory responses in PS1 KI mice. These results show hippocampal spatial memory impairments caused by the PS1 M146V mutation and age-related deterioration of the memory impairment, suggesting that PS1 KI mice are a valuable model system for the study of memory loss in AD.

Aging↗
PubMed1993

Conversion of homothallic yeast to heterothallism through HO gene disruption.

A simple method was developed for the conversion of homothallic Saccharomyces cerevisiae yeast strains to heterothallism through HO gene disruption. An integrative ho::neo disrupted allele was constructed by cloning a dominant selectable marker, the bacterial neo gene, within the HO gene. Transformation of a homothallic diploid S. cerevisiae strain with plasmid DNA containing the ho::neo allele yielded G418-resistant yeast transformants in which one of the HO alleles was replaced by the disrupted ho::neo allele. Meiotic tetrad analysis of four-spored asci from these G418-resistant transformants gave rise to haploid heterothallic and diploid homothallic tetrad progeny. The presence of the ho::neo and HO alleles in the heterothallic and homothallic progeny was confirmed by Southern-blot analysis.

Alleles↗
PubMed1987

Cloning, mapping and molecular analysis of the pyrG (orotidine-5'-phosphate decarboxylase) gene of Aspergillus nidulans.

We have modified the transformation procedures of Ballance et al. [Biochem. Biophys. Res. Commun. 112 (1983) 284-289] to give increased rates of transformation in Aspergillus nidulans. With the modified procedures we have been able to complement pyrG89, a mutation in the orotidine-5'-phosphate decarboxylase gene of A. nidulans, by transformation with a library of wild-type (wt) sequences in pBR329. We have recovered, by marker rescue from one such transformant, a plasmid (pJR15) that carries an A. nidulans sequence that complements pyrG89 efficiently. In three experiments, this plasmid gave an average of 1985 stable transformants/micrograms of transforming DNA. We have analyzed ten of these genetically and by Southern hybridization. In five transformants a single copy of the transforming plasmid had integrated at the pyrG locus, in one transformant several copies of pJR15 had integrated at this locus, in one transformant several copies of the plasmid had integrated into other sites, and in three transformants, the wt allele had apparently replaced the mutant allele with no integration of pBR329 sequences. Sequence and S1 nuclease protection analysis revealed that pJR15 contains a gene that predicts an amino acid sequence with regions of strong homology to the orotidine-5'-phosphate decarboxylases of Neurospora crassa and Saccharomyces cerevisiae. We conclude that this gene is the wt pyrG allele. Finally, we have compared the 5'- and 3'-noncoding sequences and intron splice sequences to other genes of A. nidulans and have mapped the pyrG locus to a region between the fpaB and galD loci on linkage group I.

Aspergillus nidulans↗
PubMed2001

"Mitotic drive" of expanded CTG repeats in myotonic dystrophy type 1 (DM1).

In myotonic dystrophy type 1 (DM1), an expanded CTG repeat shows repeat size instability in somatic and germ line tissues with a strong bias toward further expansion. To investigate the mechanism of this expansion bias, 29 DM1 and six normal lymphoblastoid cell lines (LBCLs) were single-cell cloned from blood cells of 18 DM1 patients and six normal subjects. In all 29 cell lines, the expanded CTG repeat alleles gradually shifted toward further expansion by "step-wise" mutations. Of these 29 cell lines, eight yielded a rapidly proliferating mutant with a gain of large repeat size that became the major allele population, eventually replacing the progenitor allele population. By mixing cell lines with different repeat expansions, we found that cells with larger CTG repeat expansion had a growth advantage over those with smaller expansions in culture. This growth advantage was attributable to increased cell proliferation mediated by Erk1,2 activation, which is negatively regulated by p21(WAF1). This phenomenon, which we designated "mitotic drive" , is a novel mechanism which can explain the expansion bias of DM1 CTG repeat instability at the tissue level, on a basis independent of the DNA-based expansion models. The lifespans of the DM1 LBCLs were significantly shorter than normal cell lines. Thus, we propose a hypothesis that DM1 LBCLs drive themselves to extinction through a process related to increased proliferation.

Cell Division↗
PubMed2004

Estimation of relative fitnesses from relative risk data and the predicted future of haemoglobin alleles S and C.

Epidemiological studies of genetic differences in disease susceptibility often estimate the relative risks (RR) of different genotypes. Here I provide an approach to calculate the relative fitnesses of different genotypes based on RR data so that population genetic approaches may be utilized with these data. Using recent RR data on human haemoglobin beta genotypes from Burkina Faso, this approach is used to predict changes in the frequency of the haemoglobin sickle-cell S and C alleles. Overall, it generally appears that allele C will quickly replace the S allele in malarial environments. Explicit population genetic predictions suggest that this replacement may occur within the next 50 generations in Burkina Faso.

Burkina Faso↗
PubMed1992

Cloning of Campylobacter jejuni genes required for leucine biosynthesis, and construction of leu-negative mutant of C. jejuni by shuttle transposon mutagenesis.

Campylobacter jejuni is a Gram-negative pathogen responsible for diarrhoeal diseases in humans. To date, very little is known about the genetic organization and molecular biology of this microorganism. The cosmid vector pHC79 was used to construct a genomic library from the total genomic DNA of C. jejuni strain C31 in Escherichia coli and recombinant cosmids capable of complementing the auxotrophic defect in leucine biosynthesis of E. coli HB101 were identified. Three of 400 clones tested were found to be capable of complementing the nutritional defect of E. coli HB101 as well as those of independent leuB mutants of E. coli strains. These results indicated that the cloned genes responsible for leucine complementation encoded an enzyme analogous to the beta-isopropylmalate dehydrogenase specified by the leuB gene in E. coli strains. The sizes of the recombinant cosmids which became stabilized in E. coli cells ranged from 12.9 to 15.4 kb compared to the expected, originally packaged, 45- to 50-kb molecules, attesting to major rearrangements occurring in this background. The recombinant plasmid pILL547 was shown to carry genes that were analogous to the leuB gene and also to the leuC and leuD genes of E. coli. The gene required for leuB complementation was subcloned on a 1.6-kb restriction fragment and was mapped more precisely by insertional mutagenesis using as transposon a newly constructed (MiniTn3-Km) element engineered to mutagenize Campylobacter genes. The leuB gene of C. jejuni was shown to be expressed from its own promoter in E. coli cells. In E. coli minicells, the cloned insert encoded a polypeptide with an apparent molecular weight of 40 kDa. A leucine auxotrophic mutant of C. jejuni strain C31 was constructed in vitro by allelic exchange, replacing the original copy of the leucine gene by an allele mutated by the insertion of the kanamycin transposable element.

Campylobacter jejuni↗
PubMed2002

Molecular background of D(C)(e) haplotypes within the white population.

BACKGROUND: D(C)(e) and D(C)e haplotypes may be encountered in the white population. Few data are available on the molecular backgrounds responsible for depressed expression of C and e. STUDY DESIGN AND METHODS: Individuals of white origin carrying a D(C)(e) genotype resulting in depressed expression of C or both C and e were subdivided into two categories based on the RBC reactivity with the human sera Mol and Hor, which contain antibodies against low-frequency antigens of the Rh (RH) system and other non-Rh low-frequency antigens. Neither Hor+, Mol+ nor Hor+, Mol- RBCs expressed the V (RH10), VS (RH20), and/or Rh32 (RH32) low-frequency antigens. These results suggested that Hor+, Mol+ variants expressed Rh33 (RH33 or Har) and FPTT (RH50), whereas Hor+, Mol- variants might express an undefined low-frequency antigen. Further serologic and molecular analyses were performed. RESULTS: Molecular analysis of Hor+, Mol+ variants revealed a hybrid gene structure RHCe-D(5)-Ce, in which exon 5 of RHCE (RHCe allele) was replaced by exon 5 of RHD (the so-called RHCeVA allele). The presence of exon 5RHD resulted in several amino acid alterations predicted in the external loop 4 of the CeVA polypeptide. Molecular analysis of Hor+, Mol- variants revealed the presence of a new RHCe allele characterized by a single point mutation C340T within exon 3 (the so-called RHCeMA allele), resulting in a R114W substitution predicted on the external loop 2 of the CeMA polypeptide. A serologic study showed a different pattern of reactivity with C and e MoAbs. CONCLUSION: Two types of mutations resulted in amino acid substitutions predicted in external loops 4 and 2, respectively, which altered both the C and e reactivity, and indicated conformation changes or defective interaction between nonadjacent loops of the Ce polypeptide. Serologic analysis showed that together with Hor and Mol sera testing, the use of different C and e MoAbs could help to identify these variants within the white population.

Alleles↗
PubMed1994

Leishmania major HEXBP deletion mutants generated by double targeted gene replacement.

The Leishmania major single-stranded DNA binding protein HEXBP contains nine 'CCHC' zinc finger motifs and binds to oligodeoxynucleotides derived from the antisense strand of the GP63 gene 5' flanking region in gel mobility shift assays and UV-crosslinking assays. In the present study a HEXBP-deficient clone of L. major was generated by double targeted gene replacement. The two HEXBP alleles were sequentially replaced with genes encoding resistance to the aminoglycoside antibiotics hygromycin B and G418 and drug-resistant clones were selected by plating on semi-solid drug-containing media. Successful deletion of both copies of the HEXBP gene implies that HEXBP is a not essential for growth of Leishmania promastigotes. Characterization HEXBP-deficient promastigotes revealed that HEXBP deficiency had no effect on the abundance of GP63 mRNA and protein in in vitro cultivated promastigotes and that HEXBP-deficient promastigotes were capable of lesion formation in BALB/c mice.

Alleles↗
PubMed2005

A high-efficiency method to replace essential genes with mutant alleles in yeast.

Temperature-sensitive (TS), internally deleted and truncated alleles are important tools to facilitate the characterization of essential genes. We have developed a straightforward method to replace a wild-type gene with a mutant allele at the endogenous locus. This method is an efficient alternative to the two-step method for integration of alleles that are compromised in function or contain multiple mutations. A strain is constructed that has the essential gene of interest disrupted by a selectable marker. Strain viability is maintained by a plasmid carrying a copy of the essential wild-type gene and the ADE3 gene. The mutant allele is cloned into an integratable vector carrying a selectable/counter-selectable marker, such as URA3. The plasmid is linearized and transformed, directing integration to the 5' or 3' region flanking the essential open reading frame (ORF). Transformants that have integrated the mutant gene at the endogenous locus can lose the autonomous plasmid carrying the wild-type copy of the essential gene and the ADE3 gene. These transformants are identifiable as white sectoring colonies, display the mutant phenotype and may be characterized. An optional second selection step on 5-fluoroorotic acid (5-FOA) selects for popouts of the integrating vector sequences, leaves the mutant allele at the endogenous locus, and recycles selectable markers. We have used this method to integrate a TS allele of SPC110 that could not be integrated by standard methods.

Alleles↗
PubMedDate not supplied

Selection for maximum longevity in mice.

In both mice and men, during the adult life span, aging causes an exponential increase in vulnerability to almost all pathologies. Thus, aging is a serious public health problem. Altering the basic mechanisms that control normal aging would be a powerful approach to reduce damage from aging processes, so research identifying these mechanisms is of vital importance. Because life spans are determined by the first biological system to malfunction, it is likely that basic mechanisms are involved in life span extension of animals already having maximum normal life spans for the species. When life spans of a species are extended, all biological systems must function for unusually long times. If there are a limited number of genes for basic mechanisms that control aging rates in multiple biological systems, then life spans can be extended relatively easily. If not, extending maximum life spans would require changes in impractically large numbers of genes, all genes involved in functional life spans of every biological system. In fact, life spans appear to increase rapidly during evolution, suggesting that changes in only a few genes are required. These genes are likely to control underlying mechanisms timing aging in multiple biological systems. The purpose of selection for increased life span is to identify these genes. An important potential problem is that all species have many defective genetic alleles that can cause early disease and death. Selection studies must be designed to distinguish between altering basic mechanisms of aging, and simply avoiding early pathologies due to defective alleles. Animal models that are short lived for their species should be avoided, because their deaths almost always result from genetic defects unrelated to mechanisms of normal aging. During selection, alleles not causing early pathologies may appear to increase life spans by replacing defective alleles in genetic regions linked to early pathologies; however, these affect early disease, not basic mechanisms of aging. A more subtle potential problem is that caloric restriction increases life spans in mice. Selection for long lived mice should focus on more basic mechanisms than breeding mice that voluntarily consume fewer calories. The fact that aging rates in different biological systems are not necessarily coordinated in different individuals suggests that normal aging is timed by more than one mechanism. Thus, the objective in selection for maximum longevity is to capture the entire set of alleles that increase longevity in a species. Wild populations are not practical to use, despite some theoretical advantages, as genes retarding aging would be confounded with those reducing the stress of captivity. Currently we use four-way crosses of inbred strains that represent maximal genetic diversity. Genetic regions important in increasing longevity will be identified using microsatellite markers distinguishing each of the four starting strains over the entire genome. Other genetic techniques proven useful for studying characteristics that are quantitatively controlled by multiple genes may also be useful in studying mechanisms timing aging; these techniques include diallele crosses, recombinant inbred lines, bilineal congenic lines and correlated genetic markers.

Aging↗
PubMed2001

Knock-in mice with a chimeric human/murine p53 gene develop normally and show wild-type p53 responses to DNA damaging agents: a new biomedical research tool.

The high prevalence and great diversity of p53 tumor suppressor gene mutations in human tumors call for development of therapeutic molecules that rescue function of aberrant p53 protein. P53 mutations also offer new approaches to the study of the origins of mutations in human cancer. An experimental mouse model with a genetically modified but normal functioning p53 gene harboring the human rather than the murine core domain, would be of considerable benefit to research on both cancer therapeutics and etiology; however, it is uncertain whether such mice would permit biological functions of p53 to be retained. Using a Cre/lox P gene-targeting approach, we have constructed a human p53 knock-in (hupki) mouse strain in which exons 4-9 of the endogenous mouse p53 allele were replaced with the homologous, normal human p53 gene sequence. The chimeric p53 allele (p53(KI)) is properly spliced, transcribed in various tissues at levels equivalent to wild-type mice, and yields cDNA with the anticipated sequence, that is, with a core domain matching that of humans. The hupki p53 protein binds to p53 consensus sequences in gel mobility shift assays and accumulates in the nucleus of hupki fibroblasts in response to UV irradiation, as is characteristic of wild-type p53. Induction of various p53-regulated genes in spleen of gamma-irradiated homozygous hupki mice (p53(KI/KI)), and the kinetics of p53-dependent apoptosis in thymocytes are similar to results with wild-type (p53(+/+)) mice, further indicating normal p53 pathway function in the hupki strain. The mice are phenotypically normal and do not develop spontaneous tumors at an early age, in contrast to knock-out (p53(-/-)) strains with a defective p53 gene. The chimeric (p53(KI)) allele thus appears to provide a biological equivalent to the endogenous murine (p53(+)) gene. This strain is a unique tool for examining in vivo spontaneous and induced mutations in human p53 gene sequences for comparison with published human tumor p53 mutation spectra. In addition, the hupki strain paves the way for mouse models in pre-clinical testing of pharmaceuticals designed to modulate DNA-binding activity of human p53.

Animals↗