The nematodes Caenorhabditis elegans and C. briggsae independently evolved self-fertile hermaphroditism from gonochoristic ancestors. C. briggsae has variably divergent orthologs of nearly all genes in the C. elegans sex determination pathway. Their functional characterization has generally relied on reverse genetic approaches, such as RNA interference and cross-species transgene rescue and more recently on deletion mutations. We have taken an unbiased forward mutagenesis approach to isolating zygotic mutations that masculinize all tissues of C. briggsae hermaphrodites. The screens identified loss-of-function mutations in the C. briggsae orthologs of tra-1, tra-2, and tra-3. The somatic and germline phenotypes of these mutations are largely identical to those of their C. elegans homologs, including the poorly understood germline feminization of tra-1(lf) males. This overall conservation of Cb-tra phenotypes is in contrast to the fem genes, with which they directly interact and which are significantly divergent in germline function. In addition, we show that in both C. briggsae and C. elegans large C-terminal truncations of TRA-1 that retain the DNA-binding domain affect sex determination more strongly than somatic gonad development. Beyond these immediate results, this collection of mutations provides an essential foundation for further comparative genetic analysis of the Caenorhabditis sex determination pathway.
Summary The development and characterisation of a new epithelial model for the experimental investigation of metastasis is described. A tissue culture cell line CMT64 was established from a spontaneous alveolar lung carcinoma of a 17 month old female C57BL/ICRF a' mouse . Subcutaneous inoculation of cells produces local tumours which give rise to a small number of lung metastases within three weeks. Four different tissue culture sublines CMT167, 170, 175 and 181 with increased metastatic ability were selected from pooled lung metastases by culture, mouse inoculation and reselection from lung metastases through four culture/inoculation cycles. These sublines are themselves heterogeneous and clones derived from them display marked differences in metastatic behaviour. Both CMT64 and its sublines have remained relatively stable in morphology and behavior since their origin, are fairly well differentiated, produce basal lamina even in metastases, and metastasise rapidly and preferentially to the lung after subcutaneous and intravenous inoculation in both syngeneic C57 and Nu/Nu mice (Franks & Layton, 1984). The expression of the metastatic potential of these cells is strongly influenced by the age and immune status of the host. The CMT64 system is a particularly useful model for experimental metastasis studies.
The cellular localization of voltage-gated calcium channels (VGCCs) and synaptic vesicle-associated proteins, SV2, synapsin I, and vesicle-associated membrane protein (VAMP) (synaptobrevin), was investigated in the guinea pig cochlea using immunocytochemistry and confocal laser scanning microscopy. Reactivity, in guinea pig, of antibodies to the alpha1 subunits of L-type, alpha1C [Cav1.2] and alpha 1D [Cav1.3]; P/Q-type, alpha1A [Cav2.1]; and R-type, a1E [Cav2.3] high voltage-activated calcium channels, was determined by Western blotting and immunolabeling of cerebellum. In the cochlea the sensory inner hair cells of the organ of Corti displayed strong intracellular staining, predominantly localized to their basolateral poles, with an antibody directed against the alpha1C subunit. Some alpha1C labeling was also observed in the inner pillar cells, in cell bodies of afferent neurons in the spiral ganglion, and in the inferior region of the spiral ligament. The supporting pillar cells were strongly immunoreactive throughout for alpha1D, but no alpha1D labeling of the inner hair cells was seen. The alpha1A subunit showed a cytoplasmic distribution in all three rows of outer hair cells. alpha1E labeling localized to the outer hair cells, predominantly in the subcuticular plate region, and also to nerve fiber bundles beneath these hair cells. Strong immunoreactivity was consistently seen with antibodies directed against SV2 and synapsin I in neuronal structures surrounding the basolateral surfaces of both the inner and outer hair cells but was absent from the sensory cells themselves. VAMP labeling was found throughout the cytoplasm of the inner hair cells and in neuronal structures beneath the hair cells. These results reveal a differential distribution of VGCC-types in the sensory and nonsensory elements of the guinea pig cochlea, with the inner hair cells expressing alpha1C L-type channels and VAMP but not synapsin I or SV2.
Little is known about the heterozygous frequency of factor VIII gene markers in the Asian Indian population. The objective of this study was to establish the heterozygous frequency of polymorphic markers within and flanking the factor VIII gene in Indians and identify those most informative for carrier screening and prenatal diagnosis. Factor VIII gene polymorphism analysis at intragenic and extragenic sites was carried out by the polymerase chain reaction (PCR) method and Southern blot procedure. Sixty-three Asian Indian haemophiliacs and their families were screened. A control group of 150 women from nonhaemophilic families were screened for two markers, HindIII and BclI. Among the intragenic markers studied, the HindIII restriction fragment length polymorphism (RFLP) showed the highest heterozygous frequency (0.52) followed by the intron 13 (0.47) and intron 22 (0. 44) short tandem repeats (STRs). Among extragenic markers, TaqI had the highest heterozygous frequency (0.75) followed by BglII (0.54). The intron 22 inversion mutation was observed in eight (40%) of 20 severe cases. In the population studied the most diagnostic polymorphisms were the intragenic markers, intron 22 (70%) STR followed by the intron 13 (52%) STR and HindIII (52%) RFLP, and the TaqI (50%) extragenic marker. Application of HindIII, BclI and the intron 22 dinucleotide repeat combined were diagnostic in 87.2% of haemophilia A families studied.
We report on a kindred segregating 2 distinct mutations of a dystrophin gene. DNA analysis showed that the second mutation, a deletion, arose in the same gene carrying the primary defect which produced a Becker phenotype in the affected males. The DNA data for this family are reported and the alternative explanations of chance occurrence and premutation are discussed to explain these unusual findings.
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