ABSTRACT
We have cloned and sequenced a mouse homologue of the human breast and ovarian cancer susceptibility gene, BRCA1. The predicted mouse Brca1 protein is composed of 1812 amino acids. The murine protein is 60% identical and 72% similar to the human BRCA1 protein. Two regions of high homology have been identified between the two proteins. First is the Cys3-His-Cys4 type zinc-finger domain that is identical between the two proteins. The second region is defined by 115 amino acids near the carboxyl end of the Brca1 protein that is 83% identical to human BRCA1 sequence. Seven of eight amino acids involved in human missense mutations that are associated with the disease were found to be conserved between the two species. In contrast, most of the amino acids that are involved in polymorphic variations were not conserved. We therefore propose that the interspecies conservation of predicted amino acid sequences can be used as an additional criterion to determine the significance of human missense mutations.
Subject(s)
Mutation , Neoplasm Proteins/genetics , Transcription Factors/genetics , Amino Acid Sequence , Animals , BRCA1 Protein , Base Sequence , Biological Evolution , Breast Neoplasms/genetics , Conserved Sequence , DNA, Complementary/isolation & purification , Humans , Mice , Mice, Inbred C57BL , Molecular Sequence Data , RNA, Messenger/metabolism , Sequence Homology, Amino AcidABSTRACT
To directly assess c-myc function in cellular proliferation, differentiation, and embryogenesis, we have used homologous recombination in embryonic stem cells to generate both heterozygous and homozygous c-myc mutant ES cell lines. The mutation is a null allele at the protein level. Mouse chimeras from seven heterozygous cell lines transmitted the mutant allele to their offspring. The analysis of embryos from two clones has shown that the mutation is lethal in homozygotes between 9.5 and 10.5 days of gestation. The embryos are generally smaller and retarded in development compared with their littermates. Pathologic abnormalities include the heart, pericardium, neural tube, and delay or failure in turning of the embryo. Heterozygous females have reduced fertility owing to embryonic resorption before 9.5 days of gestation in 14% of implanted embryos. c-Myc protein is necessary for embryonic survival beyond 10.5 days of gestation; however, it appears to be dispensable for cell division both in ES cell lines and in the embryo before that time.
Subject(s)
Cell Differentiation/genetics , Chromosome Aberrations/genetics , Embryonic and Fetal Development , Genes, myc/physiology , Proto-Oncogene Proteins c-myc/genetics , Alleles , Animals , Base Sequence , Blotting, Southern , Chromosome Disorders , DNA/analysis , Electrophoresis, Polyacrylamide Gel , Embryo, Mammalian/abnormalities , Female , Fetal Death , Genes, Lethal , Genes, myc/genetics , Heterozygote , Homozygote , Infertility/genetics , Litter Size , Male , Mice , Molecular Sequence Data , Mutagenesis, Insertional , Mutagenesis, Site-Directed , Mutation , Polymerase Chain Reaction , Proto-Oncogene Proteins c-myc/analysis , Recombination, GeneticABSTRACT
We have investigated coelectroporation as a method for introducing minor genetic changes into specific genes in embryonic stem cells. A selectable marker (neo) and a targeting replacement vector designed to insert a 4-bp insertion into exon 3 of the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were coelectroporated into embryonic stem cells and selected in G418 and 6-thioguanine (6-TG). HPRT-negative clones were obtained at a frequency of approximately 1 per 520 G418r clones. Southern analysis and the polymerase chain reaction were used to demonstrate that 3 of 36 of the 6-TG-resistant clones had the desired 4-bp insertion without any other disruption of the HPRT locus. Initial studies indicated that the other 33 6-TG-resistant clones probably resulted from the targeted integration of a concatemer containing both the targeting construct and the selectable neo gene.
Subject(s)
DNA Mutational Analysis , Transfection , Blotting, Southern , Electricity , Hypoxanthine Phosphoribosyltransferase/genetics , Recombination, Genetic , Restriction Mapping , TeratomaABSTRACT
Many experimental designs require the analysis of genomic DNA from a large number of samples. Although the polymerase chain reaction (PCR) can be used, the Southern blot is preferred for many assays because of its inherent reliability. The rapid acceptance of PCR, despite a significant rate of false positive/negative results, is partly due to the disadvantages of the sample preparation process for Southern blot analysis. We have devised a rapid protocol to extract high-molecular-weight genomic DNA from a large number of samples. It involves the use of a single 96-well tissue culture dish to carry out all the steps of the sample preparation. This, coupled with the use of a multichannel pipette, facilitates the simultaneous analysis of multiple samples. The procedure may be automated since no centrifugation, mixing, or transferring of the samples is necessary. The method has been used to screen embryonic stem cell clones for the presence of targeted mutations at the Hox-2.6 locus and to obtain data from human blood.
Subject(s)
Blotting, Southern/methods , DNA/isolation & purification , Animals , Clone Cells , DNA/blood , Genome , Humans , Mice , Restriction Mapping , Stem Cells/chemistrySubject(s)
Employer Health Costs/statistics & numerical data , Health Benefit Plans, Employee/economics , Health Maintenance Organizations/economics , Insurance Selection Bias , Cost Savings , Costs and Cost Analysis , Health Benefit Plans, Employee/statistics & numerical data , Health Maintenance Organizations/statistics & numerical data , Humans , United StatesABSTRACT
Insulin is a major regulator of lipoprotein lipase (LPL) activity. The molecular events associated with LPL regulation by insulin in 3T3-L1 adipocytes were studied by determining LPL enzyme activity, mRNA levels, protein synthetic rate, and transcription run-off activity. Adipocytes treated with insulin (10(-6) M for 48 h) had substantially higher LPL activity (mean difference compared to carrier-treated cells 146%) with little difference in LPL mRNA levels (mean level 109% of control). Insulin regulation of LPL activity was dose-dependent but changes in LPL mRNA were not. Within 2 h of hormone addition, LPL activity was higher in insulin-treated versus carrier-treated adipocytes although their LPL mRNA levels were similar. In [35S]methionine pulse-labeled adipocytes, insulin decreased LPL protein synthetic rate measured by immunoprecipitation 42-48%, although increases (75-340%) in heparin-releasable LPL activity were detected in the same cells. In contrast, during differentiation of 3T3-L1 fibroblasts to the adipocyte state, 5-80-fold increases of heparin-releasable LPL activity were closely associated with similar (8-60-fold) increases in LPL mRNA levels. LPL synthetic rate was 16-fold greater, and LPL gene transcription initiation measured by transcriptional run-off was 10-fold higher in adipocytes than in undifferentiated cells. Differentiation of 3T3-L1 fibroblasts increases transcription of the LPL gene leading to increased LPL mRNA, protein synthetic rate, and enzyme activity. Insulin regulation of LPL activity in 3T3-L1 adipocytes, however, is mediated entirely at posttranscriptional and posttranslational levels.
Subject(s)
Adipose Tissue/enzymology , Insulin/pharmacology , Lipoprotein Lipase/genetics , Protein Processing, Post-Translational/drug effects , RNA Processing, Post-Transcriptional/drug effects , Animals , Blotting, Northern , Cell Differentiation , Cells, Cultured , Kinetics , Lipoprotein Lipase/metabolism , Mice , RNA, Messenger/drug effects , RNA, Messenger/geneticsABSTRACT
Lipoprotein lipase (LPL) and hepatic lipase (HL) enzyme activities were previously reported to be regulated during development, but the underlying molecular events are unknown. In addition, little is known about LPL evolution. We cloned and sequenced a complete mouse LPL cDNA. Comparison of sequences from mouse, human, bovine, and guinea pig cDNAs indicated that the rates of evolution of mouse, human, and bovine LPL are quite low, but guinea pig LPL has evolved several times faster than the others. 32P-Labeled mouse LPL and rat HL cDNAs were used to study lipase mRNA tissue distribution and developmental regulation in the rat. Northern gel analysis revealed the presence of a single 1.87 kb HL mRNA species in liver, but not in other tissues including adrenal and ovary. A single 4.0 kb LPL mRNA species was detected in epididymal fat, heart, psoas muscle, lactating mammary gland, adrenal, lung, and ovary, but not in adult kidney, liver, intestine, or brain. Quantitative slot-blot hybridization analysis demonstrated the following relative amounts of LPL mRNA in rat tissues: adipose, 100%; heart, 94%; adrenal, 6.6%; muscle, 3.8%; lung, 3.0%; kidney, 0%; adult liver, 0%. The same quantitative analysis was used to study lipase mRNA levels during development. There was little postnatal variation in LPL mRNA in adipose tissue; maximal levels were detected at the earliest time points studied for both inguinal and epididymal fat. In heart, however, LPL mRNA was detected at low levels 6 days before birth and increased 278-fold as the animals grew to adulthood.(ABSTRACT TRUNCATED AT 250 WORDS)