Biomedical subjects

J T Mulligan

Publications and source records attributed to J T Mulligan.

4 recordsLinked to original sources

PubMed1991

Lambda YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations.

This work describes a multifunctional phage lambda expression vector system, lambda YES, designed to facilitate gene isolation from eukaryotes by complementation of Escherichia coli and Saccharomyces cerevisiae mutations. lambda YES vectors have a selection for cDNA inserts using an oligo adaptor strategy and are capable of expressing genes in both E. coli and S. cerevisiae. They also allow conversion from phage lambda to plasmid clones by using the cre-lox site-specific recombination system, referred to here as automatic subcloning. A simple method has been developed for the conversion of any plasmid into a phage lambda cDNA cloning vector with automatic subcloning capability. cDNA libraries constructed in these vectors were used to isolate genes from humans and Arabidopsis thaliana by complementation of yeast and bacterial mutations, respectively.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1989

A family of activator genes regulates expression of Rhizobium meliloti nodulation genes.

Nodulation (nod) gene expression in Rhizobium meliloti requires plant inducers and the activating protein product of the nodD gene. We have examined three genes in R. meliloti which have nodD activity and sequence homology. These three nodD genes are designated nodD1, nodD2 and nodD3, and have distinctive properties. The nodD1 gene product activates expression of the nodABC operon, as measured by a nodC-lacZ fusion or by transcript analysis, in the presence of crude seed or plant wash or the inducer, luteolin. The nodD3 gene product can cause a high basal (uninduced) level of nodC-lacZ expression and nodABC transcripts which is relatively unaffected by inducers. The effect of nodD3 is dependent on the presence of another gene, syrM (symbiotic regulator). By primer extension analysis we determined that the transcription start site is the same for nodD1 plus luteolin or nodD3-syrM mediated expression of nodA and nodH mRNAs. syrM also enhances the expression of another symbiotically important trait, production of extracellular polysaccharide. This regulatory effect of syrM requires locus syrA, which is linked to nodD3 and syrM. The syrM-syrA mediated increase in polysaccharide production requires at least some of the previously identified exo genes and may be a parallel regulatory event to the syrM-nodD3 control of nod promoters.

Blotting, Western↗

PubMed1988

Specific binding of proteins from Rhizobium meliloti cell-free extracts containing NodD to DNA sequences upstream of inducible nodulation genes.

Nodulation (nod) genes in Rhizobium meliloti are transcriptionally induced by flavonoid signal molecules, such as luteolin, produced by its symbiotic host plant, alfalfa. This induction depends on expression of nodD. Upstream of three inducible nod gene clusters, nodABC, nodFE, and nodH, is a highly conserved sequence referred to as a 'nod box.' The upstream sequences have no other obvious similarity. We have found that DNA fragments containing the regions upstream of all three inducible transcripts show altered electrophoretic mobility when treated with R. meliloti extracts. The ability of the extracts to interact specifically with these DNAs correlated with the genetic dosage of nodD1 or nodD3 and with the presence and concentration of the nodD1 or nodD3 protein (NodD1 or NodD3) in the extracts. Antiserum specific to NodD was used to construct an immunoaffinity column that permitted a substantial purification of NodD1; this preparation of NodD1 also displayed specific binding to restriction fragments containing DNA sequences found upstream of inducible nod genes. In addition, NodD-specific antiserum removed the specific DNA-binding activity from total Rhizobium cell extracts. The interaction of total extracts and of partially purified NodD protein with nod promoter sequences was competitive with an oligonucleotide representing the 3' 25-bp portion of the nod box. The interaction of R. meliloti extracts and NodD1 protein with nod gene upstream regions occurred independently of exposure of cells or extracts to flavone inducer.

Bacterial Proteins↗

PubMed1987

Transcription of Rhizobium meliloti nodulation genes. Identification of a nodD transcription initiation site in vitro and in vivo.

Nodulation genes in Rhizobium are required for invasion of the host plant. The nodABC operon is induced by plant activator molecules; this activation requires the gene product of the constitutively expressed nodD locus, which is transcribed divergently from nodABC. We are employing in vitro transcription to elucidate the molecular mechanism of nod gene activation. We used a micropurification technique to obtain RNA polymerase from Rhizobium meliloti, and here demonstrate that it initiated and terminated accurately at the Escherichia coli trp promoter-leader region. E. coli RNA polymerase, however, apparently fails to recognize R. meliloti promoters. We used the R. meliloti RNA polymerase in a minimal transcription system to attempt to localize the divergent start sites for nodD and nodABC. Transcript sizing and fingerprinting, together with synchronized single-round transcription experiments permit us to designate an in vitro transcription initiation site for nodD. Primer extension analysis of in vivo mRNA demonstrates that the initiation site which is utilized in vitro is the same site used in vivo. While nodABC is not transcribed in our minimal in vitro transcription system, this system should prove useful for the study of factors in induced cells which promote expression of this inducible promoter.

Base Sequence↗