Walter B. Dempsey

Integration host factor affects expression of two genes at the conjugal transfer origin of plasmid R100

Integration host factor (IHF) binds to two sites near the origin of transfer of the conjugative antibiotic resistance plasmid, R100. DNase I footprinting shows that one site is immediately adjacent to or/T and the gene X promoter, and another is adjacent to the traM promoter. A third site, known only from retardation gels, is near the traJ promoter. The relative promoter activities of genes X, traJ and traM are reduced in himA mutants (IHF), as measured by chloramphenicol‐resistance assays. Transcript analyses by Northern blots showed a reduction in size of the principal gene X and traJ transcripts in the absence of IHF.

Sense and antisense transcripts of traM, a conjugal transfer gene of the antibiotic resistance plasmid R100

The region of the antibiotic resistance plasmid R100 that encodes the plasmid‐specific transfer gene traM has two tandemly aligned promoters separated by 145 nucleotides. The principal transcripts are 705 and 562 nucleotides long. Minor transcripts are 1550 and 1700 nucleotides long. The 705‐base transcript appears to encode an 11 kD traM protein. The 562‐base transcript does not encode a detectable protein. When sub‐cloned on short fragments, the promoter for the 562‐base transcript initiates efficiently but that for the 705 site does not. The 3′ ends of the 705 and 562 base transcripts end inside the traJ ORF. Thus they provide additional sense RNA to compete with traJ for finP, the antisense translational regulator of traJ. A model is proposed for the participation of these sense and antisense transcripts in the control of expression of the traJ gene.

Vitamin B6 enzymes in normal and pre-eclamptic human placentae.

Abstract Placentae from normal and pre-eclamptic women were analyzed for their content of three enzymes: pyridoxal kinase, pyridoxal phosphate phosphatase, and pyridoxine phosphate oxidase. The range of values for the specific activities of the phosphatase in partially purified extracts was identical in both normal and preeclamptic placentae. The mean value was 6.4 × 10 −3 μmoles/min/mg protein for both tissues. The ranges of values for the specific activities of pyridoxal kinase and pyridoxine phosphate oxidase in the same extracts on the other hand were lower in preeclamptic placentae than in normal ones. The mean value in normal placentae was 1.52 × 10 −3 μmoles/min/mg protein for the kinase and 1.68 × 10 −5 μmoles/min/mg protein for the oxidase. In extracts of placentae from pre-eclamptic women these values were 1.23 × 10 −3 μmoles/min/mg protein and 1.29 × 10 −5 μmoles/min/mg protein respectively.

Regulation of R100 conjugation requires traM in cis to traJ

Deletion mutants of R100‐1 were constructed by classical methods to remove various segments of the traM open reading frame, pTraM‐binding sites and the traM promoters. Complementation tests showed that traM was efficiently complemented only when the frans‐acting fragment contained both the complete traM gene and the adjacent traJ promoter and leader sequences. The conclusion is that traM and traJ constitute a complex operon. A deletion mutant lacking all of the fraJ gene, and one containing a frameshifting traM deletion, retained the ability to transfer at a low level, thereby showing that neither pTraM nor pTraJ is absolutely essential for transfer.

traJ sense RNA initiates at two different proMolers in R100‐1 and forms two stable hybrids with antisense finP RNA

RNase protection experiments show that the sizes of the two R100 finP molecules are 74 and 135 nucleotides. In an RNase III mutant, finP transcripts form stable double‐stranded hybrids of 108bp and 68 bp with traJ transcripts. RNase protection experiments also show that most R100‐1 transcripts originating in traM cross the traM‐traJ intergenic region and end inside the untranslated leader region of traJ. Some extend into the traJ open reading frame. These findings mean that the antisense finP RNA, thought to regulate traJ translation, must regulate traJ transcripts from both J and M proMolers.

The finM promoter and the traM promoter are the principal promoters of the traM gene of the antibiotic resistance plasmid R100

finP multicopy repression and traJ multicopy derepression indicate that the ratio of sense to antisense transcripts is important in the regulation of R100 conjugation. The extension of R100 traM transcripts into traJ shows that promoters in traM can affect this ratio, making the regulation of traM transcription important in the regulation of R100 conjugation. Since R100 traM, traY and traI proteins bind to the traM promoter region, we examined traM transcription in R100‐1 traM, traY and traI mutants and compared it with traM transcription in both R100‐1 and R100. We verified that the traM and finM promoters provide virtually all the transcripts originating in the R100‐1 traM gene. When either is deleted, as in VAR22 or VAR30, the remaining promoter is highly active. We show here that traY positively regulates R100‐1 traM transcription, as has been found for F. We found that traI did not regulate R100‐1 traM transcription. The measured activity of the native R100 traM promoter was 12% of that in R100‐1, whereas the native R100 finM promoter was 45% of that in R100‐1. These data and data from the R100‐1 traY and traI mutants show that the activities of the two promoters varied independently.