Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

Research on genetic abnormality in the hemolytic form of hereditary elliptocytosis with homozygosity for the spectrin alpha I/74 variant

We have undertaken to identify the spectrin gene mutation in a patient with a severe hemolytic form of Hereditary Elliptocytosis with homozygosity for the spectrin alpha I/74 variant. This variant corresponds to the presence of a 74,000 peptide which is produced during mild tryptic digestion of spectrin by cleavage at the Arginine-39 of the alpha I/80,000 domain of the spectrin alpha chain (595 amino acids). We hypothesized that the alpha I/74 mutation would be closed to the cleavage site Arg-39. A genomic library built with the patient's DNA was screened with a probe corresponding to a fragment of the alpha spectrin gene. Two clones were isolated, one being of paternal, the other of maternal origin. The subclones obtained contained the alpha spectrin gene exons 2 and 3 which encode for the first 88 amino-acids of the spectrin alpha I domain. The sequences obtained did not show any abnormality. The implications of these results are discussed.     

Molecular basis and haplotyping of the alphaII domain polymorphisms of spectrin: application to the study of hereditary elliptocytosis and pyropoikilocytosis.

Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are inherited disorders of erythrocyte shape that are frequently associated with abnormalities in alpha-spectrin, one of the principal structural proteins of the erythrocyte membrane skeleton. Five polymorphisms of the alpha-spectrin gene, located in a 6-kb interval of genomic DNA, were identified and analyzed in normal and mutant alpha-spectrin alleles. Three of these polymorphisms are due to single nucleotide substitutions in the alpha-spectrin gene coding region that lead to changes in the amino acid sequence. In combination, these three polymorphisms are responsible for the different peptide phenotypes of the alphaII domain previously observed following limited tryptic digestion of spectrin protein. The most common haplotype, type 1, was found predominantly in Caucasians and was the only haplotype identified in Asians. Haplotypes 2, 3, and 4 were identified predominantly in individuals of African ancestry and were commonly found in patients with HE or HPP. Analysis of coinheritance of alphaII domain polymorphisms with alpha-spectrin gene mutations causing HE or HPP in African-American patients with HE and HPP suggests that, with one exception, a given HE/HPP mutation is present in an alpha-spectrin gene of only one haplotype, indicating a founder effect. The other two polymorphisms located in this region of the alpha-spectrin gene do not change the amino acid sequence of the encoded alpha-spectrin chain and are not in linkage disequilibrium with three of the four alphaII domain haplotypes. A model is proposed for the evolutionary origin of the different haplotypes.     

On alternatives to selection-induced mutation in the Bgl operon of Escherichia coli

Selection-induced mutations are nonrandom mutations that occur as specific and direct responses to environmental challenge. Examples of selection-induced mutations have been reported both in bacteria and in yeast. I previously showed (Hall 1988) that excisions of the mobile genetic element IS150 from within bglF are selection induced and argued that they occurred because they were potentially advantageous under the selective conditions employed. Mittler and Lenski (Mittler and Lenski 1992) have argued that such excisions are not selection induced but that they occur randomly in nondividing cells. Here I provide further evidence that IS150 excisions are induced by selection and that the excisions are immediately, rather than only potentially, advantageous to the cell. I also provide evidence that excisions, which Mittler and Lenski claim occur randomly in saturated broth cultures, actually occur after samples from those cultures are plated onto selective medium.      

Analysis of WT1 gene expression during mouse nephrogenesis in organ culture

Summary The temporal and spatial expression patterns of the Wilms tumor gene, WT1, were studied during the organogenesis of the mouse kidneyin vitro. In situ hybridization and immunocytochemistry localized cellular expression of WT1 in whole kidney organ cultures to the induced metanephric mesenchyme and developing podocytes. Organ cultures were further characterized immunocytochemically with antibodies that specifically labeled the different tubular epithelial components and supporting mesenchyme of the developing nephrons. In organ cultures, the WT1 expression pattern could be visualized in induced metanephric mesenchyme and entire cell cohorts of differentiating podocytes. Expression of WT1 and cell specific markers were retained in short-term monolayer cultures of dissociated kidneys. The development of the metanephric kidneyin vitro involves a highly restricted temporal and spatial cellular expression pattern of WT1 which closely follows that observed in tissue sections from gestational kidney isolated during organogenesis in the mouse.