The use of Y-chromosome-specific repeated DNA sequences in the analysis of testis development in an XX/XY mouse

A study, by means of Y-chromosome-specific repeated DNA probes, of mouse (ST) with small testes is reviewed. Mouse ST was shown to be a somatic mosaic of 10 % XY and 90 % XX cells. The cellular composition of the azoospermic testis reflected the overall proportions of XX and XY cells but it was found that XY cells predominated in the Sertoli cells of the testis tubules. These findings have been interpreted to indicate a fundamental role for the Sertoli cell in inducing testis organization in the indifferent gonadal rudiment, involving the expression of the Y chromosome

Microinjection of Bkm-related male-specific mouse DNA into autologous zygotes

GATA-rich Bkm-related DNA in the mouse Y chromosome is closely associated with the sex determining locus in the Sxr (sex reversed) mutation. Bkm-related male-specific DNA was gel separated from male mouse DNA as a > 23 kb band afterHue III andBst NI digestions and microinjected into mouse zygotes to determine its effects on sexual differentiation. We observed that 1 out of 7 XX embryos and 3 out of 35 weaned female mice possessed a > 23 kb male-specific band, though the hybridization intensity was very low in comparison to the normal control male. Two presumptive male carriers of the injected Bkm DNA were identified by progeny tests. Various sporadic anomalies in sexual phenotypes were noted but no obvious connection was established between these and the injected Bkm-related DNA. No significant effects on sex ratio were found

Testis development in a mouse with 10% of XY cells

Two mouse Y-chromosome-related DNA probes were used to show that an aspermic sterile male mouse with small testes contained only approximately 10% of normal XY cells dispersed among normal XX cells. Examination of testis sections by in situ hybridization revealed that XY cells were nonrandomly concentrated in the testis tubules when compared with the interstitial cell population. These observations are discussed in the context of primary sex determination and histological differentiation of the testis

Identification of colorectal cancer patients with tumors carrying the <it>TP53 </it>mutation on the codon 72 proline allele that benefited most from 5-fluorouracil (5-FU) based postoperative chemotherapy

<p>Abstract</p> <p>Background</p> <p>Although postoperative chemotherapy is widely accepted as the standard modality for Dukes' stage C or earlier stage colorectal cancer (CRC) patients, biomarkers to predict those who may benefit from the therapy have not been identified. Previous <it>in vitro </it>and clinical investigations reported that CRC patients with wild-type p53 gene (<it>TP53)</it>-tumors benefit from 5-fluorouracil (5-FU) based chemotherapy, while those with mutated <it>TP53</it>-tumors do not. However, these studies evaluated the mutation-status of <it>TP53 </it>by immunohistochemistry with or without single-strand conformation polymorphism, and the mutation frequency was different from study to study. In addition, the polymorphic status at p53 codon 72, which results in arginine or proline residues (R72P) and is thought to influence the function of the protein significantly, was not examined.</p> <p>Methods</p> <p>To evaluate the significance of the <it>TP53 </it>mutation as a molecular marker to predict the prognosis of CRC patients, especially those who received postoperative chemotherapy, we examined the mutation by direct sequencing from fresh CRC tumors and evaluated the R72P polymorphism of the mutated <it>TP53 </it>by a combined mutant allele- and polymorphic allele-specific polymerase chain reaction (PCR).</p> <p>Results</p> <p>The <it>TP53 </it>mutation occurred in 147 (70%) of 211 Japanese CRC tumors. The mutation was observed in 93 (63%) tumors on the R72 allele and in 54 (37%) tumors on the P72 allele. Although the alterations to <it>TP53 </it>have no prognostic significance for CRC patients overall, we found that Dukes' stage C CRC patients who did not receive postoperative chemotherapy and carried the mutated <it>TP53</it>-R72 showed significantly longer survival times than those with the mutated <it>TP53</it>-P72 when evaluated by overall survival (<it>p = 0.012</it>).</p> <p>Conclusion</p> <p>Using a combined mutant allele- and polymorphic allele-specific PCR, we defined the codon 72 polymorphic status of the <it>TP53 </it>mutated allele in Japanese CRC patients. We raised a possibility that Dukes' stage C colorectal cancer patients with tumors carrying <it>TP53 </it>mutation, especially the P72 allele, benefited from 5-FU based postoperative chemotherapy.</p

Rapid and Simple Detection of Hot Spot Point Mutations of Epidermal Growth Factor Receptor, BRAF, and NRAS in Cancers Using the Loop-Hybrid Mobility Shift Assay

A simple and rapid method to detect the epidermal growth factor receptor hot spot mutation L858R in lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the oligonucleotide adjacent to the mutational site, difference in electrophoretic mobility between loop-hybrids with normal and mutated DNA was distinguishable in a native polyacrylamide gel. The method was also modified to detect in-frame deletion mutations of epidermal growth factor receptor in lung adenocarcinomas. In addition, the method was adapted to detect hot spot mutations in the B-type Raf kinase (BRAF) at V600 and in a Ras-oncogene (NRAS) at Q61, the mutations commonly found in thyroid carcinomas. Our mutation detection system, designated the loop-hybrid mobility shift assay was sensitive enough to detect mutant DNA comprising 7.5% of the total DNA. As a simple and straightforward mutation detection technique, loop-hybrid mobility shift assay may be useful for the molecular diagnosis of certain types of clinical cancers. Other applications are also discussed