Biomedical subjects

R F Fisher

Publications and source records attributed to R F Fisher.

At least 19 recordsLinked to original sources

PubMed2001

Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid.

The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome. We report here the complete 1,354,226-nt sequence of pSymA. In addition to a large fraction of the genes known to be specifically involved in symbiosis, pSymA contains genes likely to be involved in nitrogen and carbon metabolism, transport, stress, and resistance responses, and other functions that give S. meliloti an advantage in its specialized niche.

Agrobacterium tumefaciens↗

PubMed2001

The composite genome of the legume symbiont Sinorhizobium meliloti.

The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

Bacterial Adhesion↗

PubMed1996

Transcription start sites for syrM and nodD3 flank an insertion sequence relic in Rhizobium meliloti.

In Rhizobium meliloti the syrM regulatory gene positively controls nod D3 and syrA, and nodD3 positively controls syrM and nod regulon genes such as nodABC, syrM and nodD3 are divergently transcribed and are separated by approximately 2.8 kb of DNA. The 885-bp SphI DNA fragment between syrM and nodD3 was subcloned and sequenced. Analysis of this intergenic region showed two open reading frames similar to those found in insertion elements of the IS3 family. We determined transcription initiation sites for both syrM and nodD3 using primer extension. The syrM transcription initiation site is 499 bp upstream of the syrM protein-coding region and downstream of a nod box which shows several differences from the R. meliloti nod box consensus sequence. We demonstrated binding of NodD3 to DNA containing the syrM nod box. The nodD3 start site maps 659 bp upstream of the nodD3 translation initiation site. A putative SyrM binding site was identified upstream of the nodD3 start site on the basis of sequence similarity to the upstream region of syrA, another locus regulated by SyrM.

Amino Acid Sequence↗

PubMed1995

A simple view of age-related changes in the shape of the lens of the human eye.

Data describing the radius of curvature of the anterior surface of the human lens have been re-examined for (i) far-accommodated lenses and (ii) near-accommodated or excised lenses. It is found that, with increasing age, the curvatures converge to an intermediate value. Taking this together with the body of work on the mechanics of accommodative lens changes, a perspective is suggested which affords a simple view of curvature changes with age. The only datum in the literature is found to fit reasonably well with this perspective.

Accommodation, Ocular↗

PubMed1993

Interactions of NodD at the nod Box: NodD binds to two distinct sites on the same face of the helix and induces a bend in the DNA.

The Rhizobium meliloti nodD gene products are positive transcriptional activators of genes required for early stages of nodule morphogenesis in the R. meliloti-alfalfa symbiosis (nod genes). The regulatory activity of NodD, a member of the LysR family of activator proteins, is mediated in part through its binding to conserved DNA sequences termed nod boxes which lie upstream of the inducible nod genes. Here we use interference footprinting to identify two NodD binding sites in the nodA, nodF and nodH nod boxes. These two binding sites are located on the same face of the DNA helix and can be separated by an additional 10 bp with retention of activity. By systematic alteration of the phasing of the two binding sites on the DNA helix, we showed that only constructs which contain both sites on the same side of the helix are recognized by NodD as determined by migration retardation assay and by in vivo activation of nod box-lacZ fusions. Moreover, NodD apparently induces a bend in the DNA upon binding at the nod box as shown by migration retardation behavior of circularly permuted nod box fragments.

Acyltransferases↗

PubMed1992

Rhizobium--plant signal exchange.

Initial stages in the Rhizobium-legume symbiosis can be thought of as a reciprocal molecular conversation: transmission of a gene inducer from legume host to bacterium, with ensuing bacterial synthesis of a morphogen that is transmitted to the plant, switching the developmental fate of the legume root. These signal molecules have a key role in determining bacterium-host specificity and the purified Nod factor compounds provide useful new tools to probe plant cell function.

Bacterial Proteins↗

PubMed1989

DNA footprint analysis of the transcriptional activator proteins NodD1 and NodD3 on inducible nod gene promoters.

The Rhizobium meliloti nodD1 and nodD3 gene products (NodD1 and NodD3) are members of the lysR-nodD gene regulator family. They are functionally distinct in that NodD1 transcriptionally activates other nod genes in the presence of a flavonoid inducer such as luteolin, while NodD3 is capable of activating nod gene expression at high levels in the absence of inducer. NodD1 and NodD3 are DNA-binding proteins which interact with DNA sequences situated upstream of the transcription initiation sites of at least three sets of inducible nod genes. We report the footprinting of NodD1- and NodD3-DNA complexes with both DNase I and the 1,10-phenanthroline-copper ion reagent. NodD1 and NodD3 both interacted with the nodABC, nodFE, and nodH promoters and protected from cleavage an extensive piece of DNA, including the nod box, from approximately -20 to -75 from the transcription start site for each of the three promoters. The constitutively activating protein NodD3 displayed an additional hypersensitive cleavage site in its footprint compared with NodD1.

Bacterial Proteins↗

PubMed1988

The mechanics of accommodation in relation to presbyopia.

The cause of presbyopia is closely related to the force of contraction of the ciliary muscle and the resistance to deformation of the crystalline lens. Two views are currently in conflict. The view of Donders (1864) that presbyopia is caused by a decrease in the force of contraction of the ciliary muscle with age, and the opposing view of Helmholtz (1855) that the lens becomes more difficult to deform with age due to lenticular sclerosis. The present paper shows that, in fact, the ciliary muscle undergoes a compensatory hypertrophy as accommodative amplitude decreases with age. The force of contraction is about 50% greater at the onset of presbyopia than in youth. However, because of increased lenticular resistance its effect on the amplitude of accommodation is small. It is shown that the reason the lens becomes more difficult to deform is not because of lenticular sclerosis, since the lens substance does not lose water. The increased difficulty of deformation is because the capsule loses its elastic force with age and the lens fibres, particularly in the nucleus, become more compacted.

Accommodation, Ocular↗

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↗

PubMed1988

The deformation matrix theory of basement membrane: a study of water flow through elastic and rigid filaments in the rat.

1. When the volume of water per unit time which flows through natural elastic basement membrane is divided by the applied pressure, the value-the hydraulic conductivity-is not constant but decreases as pressure increases. In contrast when the same membrane is tanned with glutaraldehyde and rendered inelastic, the hydraulic conductivity is constant at all pressures. 2. Over a pressure range of 0-6.7 kPa equivalent to a membrane stress of 0-195 kPa in natural elastic membrane the hydraulic conductivity (Lp) can be related by the linear equation Lp = Lp.0 + apP where P is the hydraulic pressure, Lp.0 is the initial hydraulic conductivity and ap is a constant which is the decreased hydraulic conductivity per unit pressure (correlation coefficient 0.764. P less than 0.001). 3. The initial conductivity of the basement membrane of the crystalline lens of the adult rat (lens capsule) was 47.6 +/- 7.3 x 10(-12) m s-1 Pa-1 while the decrease in hydraulic conductivity per unit increase in pressure was -3.38 x 10(-15) m s-1 Pa-2. 4. Following tanning with glutaraldehyde the hydraulic conductivity was constant at 27.4 +/- 4.0 x 10(-12) m s-1 Pa-1. 5. A change in the configuration of the superhelices of the filaments of type IV collagen which form the framework of basement membrane is termed. 'The deformation matrix theory' and can satisfactorily account for the changes in hydraulic conductivity of both natural and tanned membrane. 6. In natural membrane the filaments deform easily and the pitch of the filament superhelices is increased by axial stress induced by pressure. The filaments straighten and become compacted together and the hydraulic permeability is thereby decreased. 7. In tanned membrane the filaments become more rigid and axial stress barely deforms them: moreover the pitch of the filament superhelices is decreased so that the filaments become more closely coiled and compacted together. Because of these changes the hydraulic conductivity is reduced as compared with unstressed natural membrane and remains unaltered by increasing pressure.

Animals↗

PubMed1987

Macromolecular organization of collagen fibres in natural and tanned basement membrane.

The elastic constants and ultrastructure of natural and tanned basement membrane of the crystalline lens of the adult rat have been investigated. Sonicated and negatively stained specimens of both membranes show parallel filaments that have similar spacing of 3.5(+/- 0.1) nm and a different periodicity. In natural membrane the periodicity is 3.7(+/- 0.13) nm, whilst in tanned basement membrane the periodicity is 3.2(+/- 0.15) nm. The periodicity ratio of tanned membrane to natural membrane was 0.86 +/- 0.04, whilst the elongation ratio of tanned membrane compared with natural membrane was 0.88 +/- 0.05. In contrast to this, the thickness ratio of tanned to natural membrane was 1.098 +/- 0.045. Tanned basement membrane showed a shrinkage of 12% in length but an increase in thickness of about 10%. These data suggest, firstly, that the degree of extension of the superhelices of the filaments follows closely the degree of extension of the intact membrane and, secondly, that the coiled superhelices of tanned membrane have an angle of tilt of about 42 degrees compared with those of natural membrane, where the angle is about 50 degrees. The Young's modulus of elasticity and ultimate stress of tanned basement membrane are, respectively, eight times greater and one-third as great as natural membrane. The entropy change in basement membrane was calculated from the external work necessary to extend the tanned membrane, and was estimated to be -13.5(+/- 2.4) J K-1 mol-1. An estimate of the change in entropy from thermodynamic measurements made on a suspension of collagen tanned with glutaraldehyde was found to be -30.1(+/- 9.5) J K-1 mol-1. The two different estimates of the change in entropy of collagen following tanning suggest that in basement membrane only about 45% of the collagenous protein has an extensile helical structure.

Animals↗

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↗

PubMed1987

The influence of age on some ocular basement membranes.

(1) Changes with age in the Young's modulus of elasticity of human lens capsule and Bruch's membrane have been determined. In lens capsule the modulus decreases with age whereas in Bruch's membrane it increases. (2) Changes with age in the hydraulic conductivity of the human lens capsule and Bruch's membrane have also been determined and again show opposite changes with age. In the former membrane the hydraulic conductivity increases while in the latter it decreases. (3) The ageing changes in the respective membranes indicate that, in man, some time after 100 years in all cases the lens capsule would have a similar hydraulic conductivity to lenses with advanced cataract, while in the same subject Bruch's membrane would have lost its permeability to water.

Adult↗

PubMed1987

Maffucci's syndrome complicated by carcinoma of the breast, pituitary adenoma, and mediastinal hemangioma.

We have described a 39-year-old woman with Maffucci's syndrome, large mediastinal hemangiomas, infiltrating adenocarcinoma of the breast, and pituitary adenoma. This is the fifth reported case of Maffucci's syndrome with a coexistent pituitary adenoma, a frequency that cannot be explained by chance alone. There has been only one previously reported case in which the patient had a widened mediastinum, but the etiology was not discussed. Three of the four previously described patients with Maffucci's syndrome and a pituitary adenoma also had a proven or possible associated epithelial neoplasm. While this association is tenuous, it is considered worthy of comment.

Adenoma↗

PubMed1986

The ciliary body in accommodation.

The ciliary muscle ring contracts about 0.8 mm in radius during maximum accommodation and this change in radius does not significantly alter as the eye ages between 15 and 45 years. Despite this constant movement of the ciliary muscle ring, the force of contraction steadily increases over the same age period from 0.8 to 1.2 gms. The force of contraction of the ciliary muscle per dioptre--the myodioptre--changes both during the act of accommodation and as the eye ages. In the first case as the amplitude of accommodation increases, the myodioptre also increases in direct proportion to the amplitude. In the second case, as the eye ages an additional increase occurs at all amplitudes of accommodation. This increase is initially quite small but between the ages of 40 and 45 it becomes very much greater. As age advances this increased force of contraction is matched by a decrease in movement of the equator of the lens so that the zonule which is attached to both the ciliary body and the lens becomes increasingly stretched during accommodation and so can transmit the increased force of contraction. The zonular fibres between the ages of 15 and 45 do not appear to change in their extensile properties so the elasticity modulus remains constant with a value of 3.5 X 10(5)Nm-2. This Young's modulus is some 10 to 900 times less than the elasticity modulus respectively of lens capsule or tendon collagen but almost the same as aortic elastin.

Accommodation, Ocular↗

PubMed1985

Analysis of the requirements for transcription pausing in the tryptophan operon.

RNA polymerase pausing during transcription of the tryptophan (trp) operon leader region is postulated to be the key event that synchronizes transcription of this region with translation of the coding region for the trp leader peptide. Coupling of transcription to translation enables the cell to monitor the intracellular concentration of charged tRNATrp and determine whether polymerase should terminate transcription at the attenuator or proceed into the structural genes of the operon. We used mutant templates containing deletions of DNA segments corresponding to sequences that are predicted to form alternative RNA secondary structures to show that formation of an RNA hairpin in the leader transcript, and the concentration of the next nucleoside triphosphate to be added to the paused transcript, both markedly affect the kinetics of pausing in vitro. A model is presented that accounts for many of the findings obtained in this and other pausing studies.

Base Sequence↗

PubMed1985

Nucleotide sequence of Rhizobium meliloti 1021 nodulation genes: nodD is read divergently from nodABC.

Nodulation (nod) genes are required for Rhizobium meliloti to invade and stimulate nodule formation in its host, alfalfa. We have established the DNA sequence of nodD, nodA, and nodB, which are part of a gene cluster located 20 kb downstream of nifHDK on the R. meliloti pSym megaplasmid. The nodD open reading frame (308 amino acids) reads from proximal to nifHDK toward distal to nifHDK, divergently from nodA (196 aa) and nodB (217 aa). These two genes read from distal to nifHDK toward proximal, and are just upstream from the previously defined open reading frame for nodC. Fourteen Tn5 insertion sites have been sequenced in nodD, nodA, and nodB, revealing no major hotspots for insertion, but an overall preference for G/C bases at positions 1 and 9 of the 9-bp repeat.

Amino Acid Sequence↗

PubMed1985

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii.

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into NodR. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod mutants. All seven mutants were restored to Nod on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.

Journal Article↗