Effect of Salmonella typhimurium ferric uptake regulator (fur) mutations on iron- and pH-regulated protein synthesis.

Fur is an important regulatory protein known to function in the presence of iron as a repressor of iron-controlled genes. It was recently discovered that Fur is also essential to Salmonella typhimurium for mounting an adaptive acid tolerance response (J. W. Foster, J. Bacteriol 173:6896-6902, 1991). Because little is known about the effect of Fur on the physiology of this enteric pathogen, a systematic two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis was conducted to identify proteins whose synthesis is linked to iron levels. Mutations in the fur locus were identified and used to classify which proteins are controlled by Fur. Thirty-six proteins were overtly affected by iron availability, most of which were clearly under the control of Fur. Although most of the Fur-dependent proteins were under negative control, a significant portion (15 of 34) appeared to be under a form of positive control. Nine of the positively controlled proteins required Fur and iron for expression. However, Fur lacking iron was also required for the induction of six gene products. Surprisingly, not all iron-regulated proteins were controlled by Fur and not all Fur-dependent proteins were obviously regulated by iron status. Because fur mutants fail to mount an effective acid tolerance response, we made a comparative two-dimensional PAGE analysis of 100 total acid- and iron-regulated gene products. Production of most of these proteins was regulated by only one of the two stresses, yet a clear subset of seven genes were influenced by both acid and iron and were also controlled by fur. These proteins were also members of the acid tolerance response modulon. Consistent with the fur effect on pH-regulated protein synthesis, fur mutants lacked the inducible pH homeostasis system associated with the acid tolerance response. The results provide further evidence that Fur has an extensive impact on gene expression and cellular physiology and suggest an explanation for the acid-sensitive nature of fur mutants.     

Restricted distribution of mRNA produced from a single nucleus in hybrid myotubes

Although the proteins encoded by a single nucleus in multinucleated myotubes have a wide range of distributions within the myofiber, little is known about the distributions of their mRNAs. We have used hybrid myotubes in which one or a few nuclei are derived from myoblasts that express nonmuscle proteins to investigate this question. We find that three different mRNAs, encoding proteins that are, respectively, nuclear, cytoplasmic, and targeted to the ER, have similar distributions within myotubes. Each is confined to an area within approximately 100 microns of the nucleus that expresses it.     

A single amino acid mutation in CDR3 of the 3-14-9 L chain abolished expression of the IDA 10-defined idiotope and antigen binding.

The ABPC 48 myeloma protein and the 3-14-9 mAb derive their V region genes from the same VH and V kappa gene families. They also share a cross-reactive idiotope defined by the anti-Id mAb IDA 10. Whereas ABPC 48 is specific for bacterial levan, 3-14-9 showed a significant Ag-binding activity to aminophenyl-beta-N-acetylglucosaminide (AZO). In order to define the molecular basis of idiotope expression and Ag-binding activity, we have cloned the genes encoding the 3-14-9 H and L chain V region genes, generated antibodies that carry mutations within the L chain genes, by site-directed mutagenesis, and investigated the effects of those mutations with respect to IDA 10 idiotope expression and binding to AZO. Our findings show that, whereas expression of the IDA 10-defined idiotope requires association of both the H and L chains, a single change (glycine to phenylalanine) at position 91 in the third complementarity-determining region of the L chain abolished both idiotope expression and Ag-binding activity. In addition, a L chain change of alanine to threonine at position 25 allowed idiotope expression to some extent but significantly reduced binding activity to AZO. These data suggest that a single amino acid change can play a crucial role in the functional activity and structural integrity of antibodies.     

Asymmetric somatic cell hybridization in plants

As part of an investigation into whether it would be possible to use UV radiation as a suitable pretreatment of the donor cells in asymmetric hybridization experiments, the effects of this treatment on sugarbeet (Beta vulgaris L.) protoplast DNA have been determined and compared with those of gamma radiation. Both nuclear and mitochondrial DNAs have been examined. The dose ranges chosen had previously been determined to be potentially applicable for fusion experiments. Pulsed field gel electrophoresis and standard agarose gel electrophoresis have been used in combination with laser scanning densitometry to gain an insight into the precise nature and degree of DNA damage resulting from irradiation. It was observed that UV radiation introduced substantial modifications to sugarbeet DNA. Double-strand breaks were detected, the number of which was found to be directly proportional to the dose applied. Such breaks indicate that UV radiation results in substantial chromosome/chromatid fragmentation in these cells. Chemical modifications to the DNA structure could be revealed by a significant reduction in DNA hybridization to specific mitochondrial and nuclear DNA probes. Following gamma irradiation at equivalent biological doses (i.e. those just sufficient to prevent colony formation) much less damage was detected. Fewer DNA fragments were produced indicating the presence of fewer double-strand breaks in the DNA structure. In comparison to UV treatments, DNA hybridization to specific probes following gamma radiation was inhibited less. For both treatments, mitochondrial DNA appeared more sensitive to damage than nuclear DNA. The possibility that DNA repair processes might account for these differences has also been investigated. Results indicate either that repair processes are not involved in the effects observed or that DNA repair occurs so fast that it was not possible to demonstrate such involvement with the experimental system used. The general relevance of such processes to asymmetric cell hybridization is discussed.     

DNA radiation damage and asymmetric somatic hybridization: Is UV a potential substitute or supplement to ionising radiation in fusion experiments?

Recent reports have revealed that the asymmetric nature of the nuclear genome of somatic hybrids, produced following the irradiation of one of the parents with X- or gamma rays, is generally much less than had been anticipated. As a consequence, we have begun to investigate whether UV radiation might be used as an alternative or indeed a supplement to the presently-used ionising radiation techniques in such experiments. Cell culture studies have revealed that UV radiation induces the desired physiological effects in sugar beet ( Beta vulgaris ) protoplasts, namely, a prevention of cell division without immediate cytotoxicity. Preliminary studies using denaturing and pulsed field gel electrophoresis have shown that UV can also induce substantial physical fragmentation of DNA. When using the same techniques, less breakdown was observed following gamma radiation. All results were highly reproducible. Such results augur well for the potential use of UV in asymmetric somatic cell fusion experiments.     

The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.

The function of rac, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, rac1 rapidly stimulated actin filament accumulation at the plasma membrane, forming membrane ruffles. Several growth factors and activated H-ras also induced membrane ruffling, and this response was prevented by a dominant inhibitory mutant rac protein, N17rac1. This suggests that endogenous rac proteins are required for growth factor-induced membrane ruffling. In addition to membrane ruffling, a later response to both rac1 microinjection and some growth factors was the formation of actin stress fibers, a process requiring endogenous rho proteins. Using N17rac1 we have shown that these growth factors act through rac to stimulate this rho-dependent response. We propose that rac and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin.     

The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette.

The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.     

A genetic linkage map of human chromosome 5 with 60 RFLP loci.

A genetic map of human chromosome 5 that contains 60 restriction fragment length polymorphism (RFLP) loci in one linkage group has been constructed. Segregation data using these markers and 40 large multigenerational families supplied by the Centre d'Etude du Polymorphisme Humain have been collected. Linkage analyses were performed with the program package CRI-MAP; using odds greater than 1000:1, 30 RFLP loci could be placed on the map. This genetic map spans 289 cM sex-equal, 353 cM in females, and 244 cM in males. While the relative rate of recombination for female meioses is nearly twice that of males over much of the chromosome, several instances of statistically significant excess male recombination were observed. The order of probes on the genetic map has been confirmed by their physical order as determined by somatic cell hybrid lines containing deletions of normal chromosome 5. There is concordance between the physical positions of markers and their genetic positions. Our most distal probes on the genetic map are cytologically localized to the most distal portions of the chromosome. This suggests that our genetic map spans most of chromosome 5.