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Notes: The authors investigated expression of BRCA1 from alternative transcripts possessing different 5' UTRs. RT-PCR using Promega's AMV reverse transcriptase was performed to generate cDNAs from total RNA isoated from human tissue. Promega's Taq DNA Polymerase was used for the PCR reaction. The authors also prepared two mRNAs that contained one or the other of the 2 alternative 5' UTRs (ex1a or ex1b) fused with the luciferase coding region. To generate the corresponding DNA for these constructs, the luciferase coding region was amplified from Promega's pGEM vector and ligated to PCR amplified ex1a or exlb. These ligation products served as the template amplifying two cDNA constructs (exla-luc or ex1b-luc).Ten additional cDNAs were made containing a variety of changes to the 5'UTR region of BRCA1. In vitro translation experiments were performed to determine how the composition of the 5'UTR affected protein expression levels (using Promega's RNasin to protect transcribed mRNA; T7 polymerase buffer; and rabbit reticulocyte and wheat germ extracts for translation). The authors conclude that secondary structures associated with elements in the longer 5'UTR reduced translation rates and could be responsible for the reduced expression of BRCA1 often associated with spontaneous ovarian and breast cancers. (2441)

Notes: Several different gap junction channel subunit isotypes, also known as connexins, were synthesized in vitro in the presence of Canine Microsomal Membranes. Translation reactions were spun through a sucrose cushion to separate the membrane fraction from the supernatant. Connexins that integrated into the microsomes formed homo- and heterooligomeric structures. The assembled proteins in the microsome were fused with a synthetic membrane and single channel conductance was measured and found to be very similar to wild-type membranes. (1618)

Notes: The paper looked at the insertion of a model protein with one, two and four transmembrane segments and different distributions of positively charged residues in the N-terminal tail and the polar loops. The proteins were translated in the presence of the microsomes. Translocation of polypeptides to the lumenal side of the microsomes was assayed by prevention of N-linked glycosylation through competitive inhibition by the addition of a glycosylation acceptor tripeptide but not by a nonacceptor tripeptide and by proteinase K treatment of the microsomes. All techniques are referenced. (2033)

Notes: The RSP2 protein was in vitro translated using Promega's Rabbit Reticulocyte Lysate in the presence of microsomes. The protein was not protected from proteinase K digestion and deemed to be a cytoplasmic protein. (1626)

Notes: The protein with six membrane-spanning domains was translated using Rabbit Reticulocyte Lysate in the presence of Canine Microsomal Membranes. The protein was N-glycosylated and this glycosylation was sensitive to endoglycosidase H. Mutants containing one to all six transmembrane domain were constructed and immunoprecipitates tested for susceptibility to protease digestion and determination of membrane topology. The full protein could only be inserted cotranslationally since incubation of translation products with puromycin and the membranes produced no protection from the proteases. (1648)

Notes: The Protein Truncation Test (PTT) is investigated as a rapid, first mutation screening approach in exon 11 of the BRCA1 gene. Eighty-one percent of the mutations found in 63 patients are truncation mutations, and 49% of these mutations are in exon 11 so the PTT is a sound screening method. The Wheat Germ Extract performed better than the Rabbit Reticulocyte Lysates with the primer set used. The investigators suggest that the choice of translation be empirically determined for each primer set used. (0538)