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Biomedical subjects

J Beckwith

Publications and source records attributed to J Beckwith.

At least 19 recordsLinked to original sources
PubMed1992

Assembly of a hetero-oligomeric membrane protein complex.

The maltose transporter of Escherichia coli is a hetero-oligomeric complex located in the cytoplasmic membrane of the cell. The in vivo assembly of this complex has been examined by using an assay based on the proteolytic sensitivity of one of its components, MalF. Immediately after synthesis and insertion into the membrane, MalF is sensitive to exogenously added proteases. In a time- and complex assembly-dependent fashion, MalF becomes protease resistant. Using this assay, we show that MalF is inserted into the membrane independently of other components of the transport complex. The assembly of the maltose transport complex occurs subsequently from a pool of freely diffusing protein in the membrane. This assembly process is efficient and occurs with rapid kinetics.

ATP-Binding Cassette Transporters↗
PubMed1992

The dynamics of assembly of a cytoplasmic membrane protein in Escherichia coli.

The topology of integral cytoplasmic membrane proteins can be analyzed using alkaline phosphatase fusions by determining which constructs have low and which have high specific activity. We show that in all cases the enzymatic activity is due to the fraction of the alkaline phosphatase moiety of the fusion protein localized to the periplasm. We present evidence that these fusions can also be used to analyze the process of assembly of cytoplasmic proteins into the membrane. The rate of acquisition of protease resistance of the alkaline phosphatase moiety of such hybrid proteins is compared for fusions to periplasmic and cytoplasmic domains. We show that this process, which is assumed to be representative of export of alkaline phosphatase, is significantly slower for fusions to cytoplasmic and certain periplasmic domains than for most periplasmic domains. These results are discussed in the context of the normal assembly of integral membrane proteins.

ATP-Binding Cassette Transporters↗
PubMed1992

Genetic testing in the workplace: a view from the USA.

Progress in human genetics, accelerated by the Human Genome Project, is leading to a rapid proliferation in the number of genetic tests. Although they have their benefits, genetic tests have in the past been used by various societal institutions, including employers, to discriminate against and stigmatize individuals. In the light of the increase in the number of tests and some current reports of discrimination in employment, a variety of legal and regulatory measures have been proposed to prevent genetic discrimination in the workplace.

Genetic Diseases, Inborn↗
PubMed1992

The genetic analysis of human behavior: a new era?

Recent developments in DNA-based techniques may revolutionize the study of human behavioral genetics. However, unless these methods are used with great care, many of the same mistakes which have plagued non-molecular genetic analyses of behavior will reoccur. Errors in the application of genetic approaches and in the interpretation of results have been a common feature of published studies in this field. We review studies in human behavioral genetics, focusing on those using identical twins and DNA-based linkage techniques in order to draw attention to recurrent problems in molecular and non-molecular studies. We suggest possible guidelines for future research in the area of the biological basis of human behavior.

Adoption↗
PubMed1992

Protein export in Escherichia coli.

The export of protein from Escherichia coli has been studied by genetic, biochemical and biophysical techniques. These studies have defined a number of steps in the export pathway and have identified the cellular components required for the translocation process. New information is presented on the function of some of these components.

Bacterial Proteins↗
PubMed1992

FtsL, an essential cytoplasmic membrane protein involved in cell division in Escherichia coli.

We have identified a gene involved in bacterial cell division, located immediately upstream of the ftsI gene in the min 2 region of the Escherichia coli chromosome. This gene, which we named ftsL, was detected through characterization of TnphoA insertions in a plasmid containing this chromosomal region. TnphoA topological analysis and fractionation of alkaline phosphatase fusion proteins indicated that the ftsL gene product is a 13.6-kDa cytoplasmic membrane protein with a cytoplasmic amino terminus, a single membrane-spanning segment, and a periplasmic carboxy terminus. The ftsL gene is essential for cell growth and division. A null mutation in ftsL resulted in inhibition of cell division, formation of long, nonseptate filaments, ultimate cessation of growth, and lysis. Under certain growth conditions, depletion of FtsL or expression of the largest ftsL-phoA fusion produced a variety of cell morphologies, including Y-shaped bacteria, indicating a possible general weakening of the cell wall. The FtsL protein is estimated to be present at about 20 to 40 copies per cell. The periplasmic domain of the protein displays a sequence with features characteristic of leucine zippers, which are involved in protein dimerization.

Amino Acid Sequence↗
PubMed1992

Discrimination as a consequence of genetic testing.

Genetic discrimination refers to discrimination directed against an individual or family based solely on an apparent or perceived genetic variation from the "normal" human genotype. We describe here the results of a case history study designed to assess whether or not genetic discrimination exists. Using the above definition of genetic discrimination and applying stringent criteria for case selection, we find that genetic discrimination exists and is manifested in many social institutions, especially in the health and life insurance industries. Stigmatization, and denial of services or entitlements to individuals who have a genetic diagnosis but who are asymptomatic or who will never become significantly impaired, is noted. Follow-up comprehensive studies on the significance and varieties of genetic discrimination are needed. In order to avoid creating a new social underclass based on genetic discrimination (the "asymptomatic ill"), existing and future genetic testing or screening programs need review by medical, scientific, legal, and social policy experts, as well as the public, and may require modification.

Adult↗
PubMed1991

Membrane insertion of the Escherichia coli MalF protein in cells with impaired secretion machinery.

The MalF protein is an integral membrane protein of Escherichia coli containing eight membrane-spanning stretches and a large periplasmic domain of approximately 180 amino acids. We have asked whether this protein is dependent for its membrane insertion on the bacterial secretion machinery specified by the sec genes. Using azide to inhibit the SecA protein and sec mutants to reduce the functioning of the machinery, we have studied the membrane assembly of MalF and beta-galactosidase and alkaline phosphatase fusions to MalF. In no case did we see an effect of reducing sec gene function on the insertion of MalF or fusion proteins. Selection for mutants that would cause internalization of a MalF-beta-galactosidase hybrid protein yielded no mutations in sec genes. Our results suggest that MalF can assemble in the membrane independently of the bacterial secretion machinery.

ATP-Binding Cassette Transporters↗
PubMed1991

Identification of a protein required for disulfide bond formation in vivo.

We describe a mutation (dsbA) that renders Escherichia coli severely defective in disulfide bond formation. In dsbA mutant cells, pulse-labeled beta-lactamase, alkaline phosphatase, and OmpA are secreted but largely lack disulfide bonds. These disulfideless proteins may represent in vivo folding intermediates, since they are protease sensitive and chase slowly into stable oxidized forms. The dsbA gene codes for a 21,000 Mr periplasmic protein containing the sequence cys-pro-his-cys, which resembles the active sites of certain disulfide oxidoreductases. The purified DsbA protein is capable of reducing the disulfide bonds of insulin, an activity that it shares with these disulfide oxidoreductases. Our results suggest that disulfide bond formation is facilitated by DsbA in vivo.

Amino Acid Sequence↗
PubMed1991

Proper insertion of a complex membrane protein in the absence of its amino-terminal export signal.

The MalF protein spans the Escherichia coli cytoplasmic membrane eight times. Deletion of the first transmembrane stretch of MalF, which acts as an export signal, results in a truncated protein that still exhibits high levels of maltose transport activity. These and additional results indicate that the orientation of a membrane protein is not determined by the amino-terminal export signal, topological information is distributed throughout the MalF protein, and insertion of a protein into the cytoplasmic membrane can occur nonsequentially.

ATP-Binding Cassette Transporters↗
PubMed1991

"Sequence-gazing?".

Explore the source record for details and available documents.

Bacterial Proteins↗
PubMed1991

One of three transmembrane stretches is sufficient for the functioning of the SecE protein, a membrane component of the E. coli secretion machinery.

The E. coli secE (prlG) gene codes for an integral cytoplasmic membrane protein which is part of the cell's secretory machinery. A deletion of nearly the entire gene renders the cell dependent on the presence of a complementing secE+ plasmid, indicating that the SecE protein is essential for growth. Deletions which remove carboxy-terminal sequences or substantial amounts near the amino-terminus of SecE can still complement the lethal deletion. This deletion analysis suggests that the essential domain of the SecE protein includes only a single one of its three hydrophobic membrane-spanning segments. Two of three dominant prlG signal sequence suppressors map to this segment. Consistent with the insensitivity of SecE to major structural changes, several cold-sensitive mutations cause lethality not because of any change in the protein, but because of a reduction in its level of expression. Our results suggest that higher levels of the protein are needed at the lower temperature. These findings are discussed in terms of the interactions between various components of the secretory machinery.

Alleles↗
PubMed1991

Decoding signals for membrane protein assembly using alkaline phosphatase fusions.

We have used genetic methods to investigate the role of the different domains of a bacterial cytoplasmic membrane protein, MalF, in determining its topology. This was done by analyzing the effects of MalF topology of deleting various domains of the protein using MalF-alkaline phosphatase fusion proteins. Our results show that the cytoplasmic domains of the protein are the pre-eminent topogenic signals. These domains contain information that determines their cytoplasmic location and, thus, the orientation of the membrane spanning segments surrounding them. Periplasmic domains do not appear to have equivalent information specifying their location and membrane spanning segments do not contain information defining their orientation in the membrane. The strength of cytoplasmic domains as topogenic signals varies, correlated with the density of positively charged amino acids within them.

ATP-Binding Cassette Transporters↗
PubMed1991

Amyotrophic lateral sclerosis and life-sustaining therapy: patients' desires for information, participation in decision making, and life-sustaining therapy.

To identify the wishes of patients with amyotrophic lateral sclerosis (ALS) for information, participation in decision making, and life-sustaining therapy and to determine whether these wishes are stable over time, we conducted a prospective survey (baseline and 6-month follow-up interviews) of 38 consecutive patients with an established diagnosis of ALS at the University of Chicago Motor Neuron Disease Clinic. Demographic data, clinical stage of ALS, illness experience, wishes for information, and desires for participating in decisions about life-sustaining therapy were elicited. Patients readily expressed their wishes for specific information on communication aids and ventilator care for respiratory failure. Demographic, socioeconomic, and clinical characteristics did not predict patients' desires for information and decision making. The preferences for information and participation in decisions were stable during the 6-month study period, whereas preferences for cardiopulmonary resuscitation in two hypothetical circumstances were less stable. Changes were unrelated to demographic or clinical characteristics of the patients. Because many patients with ALS change their preferences for life-sustaining therapy, advance directives for end-of-life care must be reevaluated periodically.

Adult↗
PubMed1991

Escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm.

The cysteines of the Escherichia coli periplasmic enzyme alkaline phosphatase, which are involved in disulfide bonds in the native enzyme, were found to be fully reduced when the protein was retained in the cytoplasm. Under these circumstances the cysteines remained reduced for at least several minutes after the synthesis of the protein was completed. This contrasted with the normally exported protein, wherein disulfide bonds formed rapidly. Disulfide bond formation accompanied export and processing. The implications of these findings for the inactivity of the enzyme in the cytoplasm are discussed.

Alkaline Phosphatase↗
PubMed1991

The FtsQ protein of Escherichia coli: membrane topology, abundance, and cell division phenotypes due to overproduction and insertion mutations.

The ftsQ gene is one of several genes thought to be specifically required for septum formation in Escherichia coli. Published work on the cell division behavior of ftsQ temperature-sensitive mutants suggested that the FtsQ product is required throughout the whole process of septum formation. Here we provide additional support for this hypothesis based on microscopic observations of the cell division defects resulting from insertional and temperature-sensitive mutations in the ftsQ gene, and constitutive overexpression of its gene product. On the basis of the published, predicted amino acid sequence of the FtsQ protein and our analysis of fusion proteins of the FtsQ protein to bacterial alkaline phosphatase, we conclude that FtsQ is a simple cytoplasmic membrane protein with a approximately 25-amino-acid cytoplasmic domain and a approximately 225-amino-acid periplasmic domain. We estimate that the FtsQ protein is present at about 22 copies per cell.

Bacterial Proteins↗