RNA destabilization by the granulocyte colony-stimulating factor stem-loop destabilizing element involves a single stem-loop that promotes deadenylation.

Granulocyte colony-stimulating factor (G-CSF) mRNA contains two distinct types of cis-acting mRNA destabilizing elements in the 3'-untranslated region. In addition to several copies of the AU-rich element the G-CSF mRNA also contains a destabilizing region that includes several predicted stem-loop structures. We report here that the destabilizing activity resides in a single stem-loop structure within this region. A consensus sequence for the active structure has been derived by site-directed mutagenesis, revealing that a three-base loop of sequence YAU and unpaired bases either side of the stem contribute to the activity. The helical nature of the stem is essential and the stem must be less than 11 bp in length, but the destabilizing activity is relatively insensitive to the sequence within the helix. The stem-loop increases the rate of mRNA deadenylation, most likely by enhancing the processivity of the deadenylation reaction. A protein that binds the stem-loop, but not an inactive mutant form, has been detected in cytoplasmic lysates.

Precise sex selected births of mice following single cell embryo biopsy and Y-linked testis-specific gene analysis.

We report on the birth of 39 mice following single cell embryo biopsy and precise sex determination following in-vitro fertilization. Polymerase chain reaction was used to amplify fragments of the mouse testis-specific gene sequence (pYMT2/B) on the Y chromosome and the ovary-specific gene (ZP3) sequence on chromosome 5 from the single biopsied cell. Embryo biopsy was not associated with any deleterious effects.

Analysis of the 18S ribosomal RNA gene of Strongyloides stercoralis.

The entire 1766 bases of the 18S rRNA gene of Strongyloides stercoralis have been sequenced. The gene has a 38% G+C content. Although it is similar in length to the 18S rRNA gene of Caenorhabditis elegans, the only other completely sequenced nematode 18S rRNA gene, it is only 69% identical. Closely related helminths will need to be sequenced in order to delineate sequences specific for the diagnosis of strongyloidiasis.

Investigation of spontaneously aborted concepti for microbial DNA: investigation for cytomegalovirus DNA using polymerase chain reaction.

Karyotypic analysis of concepti spontaneously aborted in the first trimester shows approximately 50% of these concepti contain abnormal chromosomes. In order to determine whether cytomegalovirus (CMV) infection may play a role in the early loss of pregnancies with either normal or abnormal chromosomes, we have developed an assay to amplify CMV DNA in DNA extracts from spontaneously aborted concepti using the polymerase chain reaction (PCR). Using PCR, we were unable to detect CMV DNA in any of 350 spontaneously aborted concepti. Viral cultures were also negative when 36 of these were tested. Our results suggest that CMV infection is an unlikely cause of pregnancy loss in the first trimester of pregnancy.

Autobac susceptibility testing of methicillin-resistant Staphylococcus aureus isolated in an Australian hospital.

Semiautomated rapid broth elution (Autobac Multi-Test System; General Diagnostics, Div. Warner-Lambert Co., Morris Plains, N.J.) and disk diffusion tests were compared with an agar dilution breakpoint method to determine the antibiotic susceptibility of 147 methicillin-resistant Staphylococcus aureus isolates from our hospital. Although the disk diffusion method, in general, correlated well with the agar dilution tests, the overall agreement of the Autobac tests with agar dilution tests was only 79%, with many very major discrepancies occurring with clindamycin (88%), gentamicin (33%), and methicillin (15%). When we used a 10-fold higher inoculum for the Autobac tests, all isolates were shown to be resistant to methicillin, but significant numbers of major and minor discrepancies occurred with chloramphenicol, fusidic acid, and neomycin. The majority of isolates were shown to belong to three biotypes, distinguishable by lactose fermentation, lipolysis, hemolysis, and pigment production. The antibiotic susceptibility profile of one biotype was found to be markedly different from those of the other biotypes and contained a high incidence of clindamycin susceptibility and neomycin, gentamicin, and kanamycin resistance. In contrast, the other two biotypes had a high incidence of clindamycin, gentamicin, and kanamycin resistance and neomycin susceptibility and accounted for most of the very major discrepancies in the clindamycin and aminoglycoside tests. In these methicillin-resistant S. aureus strains, discrepancies possibly may arise from partial expression of methicillin resistance, dissociated or inducible clindamycin resistance, and instability of gentamicin resistance.