Mutant beta-spectrin 4 causes auditory and motor neuropathies in quivering mice.

The autosomal recessive mouse mutation quivering (qv), which arose spontaneously in 1953, produces progressive ataxia with hind limb paralysis, deafness and tremor. Six additional spontaneous alleles, qvJ, qv2J, qv3J, qv4J, qvlnd and qvlnd2J, have been identified. Ear twitch responses (Preyer's reflex) to sound are absent in homozygous qv/qv mice, although cochlear morphology seems normal and cochlear potentials recorded at the round window are no different from those of control mice. However, responses from brainstem auditory nuclei show abnormal transmission of auditory information, indicating that, in contrast to the many known mutations causing deafness originating in the cochlea, deafness in qv is central in origin. Here we report that quivering mice carry loss-of-function mutations in the mouse beta-spectrin 4 gene (Spnb4) that cause alterations in ion channel localization in myelinated nerves; this provides a rationale for the auditory and motor neuropathies of these mice.

A comparison of plasma cystatin C and plasma creatinine for the screening of renal function in lithium-treated patients.

BACKGROUND: Renal insufficiency is a serious complication of lithium treatment. Therefore, regular monitoring of plasma (P) creatinine is always part of lithium treatment safety routines. Recently P-cystatin C-estimated glomerular filtration rate (cystatin C-eGFR) had been launched as a preferable alternative to P-creatinine. AIMS: To find out which of the two alternatives to prefer in the safety routines for lithium-treated patients. MATERIAL: All 201 patients on lithium treatment at the Department of Psychiatry in Lund, Sweden. METHODS: During 14 months P-cystatin C was included in the safety routines besides routine P-creatinine every 4 months. At the end of the study period, 182 patients were eligible for analysis. With iohexol clearance as reference for GFR (performed in 111/182 patients) we calculated positive and negative predictive values (PPV, NPV) for P-creatinine and for creatinine-eGFR and cystatin C-eGFR, obtained by prediction equations. We also calculated the agreement between the measures of GFR (including repeatability). RESULTS: PPV for cystatin C-eGFR (65%) was better than for creatinine-eGFR (48%). Combining the two resulted in a PPV of 56% and marginally increased NPV to 95%. The average of cystatin C-eGFR and creatinine-eGFR yielded PPV 67% and NPV 92%. The agreement between creatinine-eGFR and GFR was better than the agreement between cystatin C-eGFR and GFR, but both were clinically unacceptable. The repeatability of P-creatinine was acceptable for psychiatric purposes. The repeatability of cystatin C-eGFR was inferior to that of P-creatinine. CONCLUSION: Our results do not justify replacing P-creatinine by cystatin C-eGFR in the lithium treatment safety routines.

Feedback regulation of rRNA synthesis in Escherichia coli. Requirement for initiation factor IF2.

It has been shown that the transcription of rRNA in Escherichia coli is feedback-regulated by its own transcription products through a negative feedback loop which appears to require the assembly of rRNA into complete ribosomes. In order to examine whether the feedback loop involves the ribosomes' main function, translation, we have constructed a strain in which the chromosomal copy of infB, encoding IF2, was placed under lac promoter/operator control, and the effects of limitation of translation initiation factor IF2 on the regulation were examined. By varying the concentration of a lac operon inducer, isopropyl thiogalactoside (IPTG), it was possible to vary the cellular concentration of IF2. Under the growth conditions used, decreasing the concentration of IF2 about twofold affected the growth rate only slightly, but further deprivation of IF2 resulted in a significant decrease in growth rate, an increase in RNA content and a large accumulation of non-translating ribosomes. These accumulated ribosomes were apparently unable to cause feedback regulation of rRNA synthesis in the absence of sufficient IF2. When a higher concentration of IPTG was added to these IF2-deficient cells, a rapid increase in the IF2 level and a significant decrease in the rate of RNA accumulation were observed before the new steady-state growth was attained. These results indicate that IF2 apparently is necessary for feedback regulation of stable RNA and imply that ribosomes must enter translation for feedback regulation to occur.

Microsatellite marker panels for use in high-throughput genotyping of mouse crosses.

Several microsatellite genotyping panel sets have been developed that are polymorphic between C57BL/6J and CAST/Ei mice, or C57BL/6J and DBA/2J. One set of markers for each strain pair has an intermarker distance of approximately 20 cM, and a second set has an intermarker distance of 5 cM. The 20-cM set contains 105 markers for C57BL/6J x DBA/2J and 108 for C57BL/6J x CAST/Ei, divided into 13 panels. Each 5-cM set includes 350 markers arranged into 45 panels. A panel contains a number of primer pairs whose fluorescently labeled PCR products can be pooled together and separated on one lane of a polyacrylamide gel. The sets are arranged by the size of the PCR product and by the type of fluorescent dye; 5-cM sets are also arranged by chromosomal region. The 20-cM sets are most useful for full-genome scans, the 5-cM sets are useful for full-genome and/or for region-specific chromosome screens. Both sets were proven as useful tools for speed congenic development, quantitative trait loci (QTL) analysis and physical mapping. These panel sets provide a throughput of 1,536-2,304 mouse genotypes daily per one gel-based system. Whole genome scans of one animal require 13 or 48 gel lanes, with 20 cM or 5 cM density, respectively.

Two Drosophila learning mutants, dunce and rutabaga, provide evidence of a maternal role for cAMP on embryogenesis.

The dunce gene of Drosophila melanogaster encodes a cAMP-specific phosphodiesterase (form II). Mutant dunce flies have elevated levels of cAMP and exhibit a number of defects including learning deficiencies and female sterility. Two partial suppressors of the female sterility phenotype have been selected in an X chromosome containing a dunce null mutation. Both suppressors are associated with reduced AC2 activity. Complementation analyses suggest that both are alleles of the learning mutant rutabaga. Females homozygous for dunce null mutations that abolish PDE activity do not deposit eggs. The suppressors exhibit differential effects on egg deposition and production of progeny; double-mutant females deposit many eggs that fail to hatch, but some develop to adults. These adult progeny exhibit morphological defects that are confined mostly to the second and third thoracic segments or to the first five abdominal segments. These observations demonstrate that the dunce gene is required in adult females for egg laying and that the dunce gene provides an essential maternal function required for normal development of the zygote. Clonal analysis, employing the dominant female-sterile mutation ovoD1, demonstrates that the former requirement for PDE activity resides in somatic cells and that the latter requirement resides in germ line cells. Female germ line cells homozygous for a dunce null mutation produce oocytes that fail to develop. Thus, homozygous dunce null-mutant zygotes develop to adults solely because of the enzyme or mRNA present in the oocytes of heterozygous mothers. Mutant alleles of rutabaga act in the germ line cells to partially suppress the developmental defects caused by dunce mutations. Thus the rutabaga gene, as well as the dunce gene, functions in both somatic and germ line cells.

Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation in Escherichia coli.

To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 in Escherichia coli, the infC, rpmI and rpIT genes encoding IF3, L35 and L20, respectively, were placed under the control of lac promotor/operator sequences. Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium. Lysogenic strains were constructed with recombinant lambda phages that express either rpmI and rplT or infC and prmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations. At low cellular concentration of IF3, but not L20, decreases and the growth rate slows. Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo. During slow growth, the ratio of RNA to protein increases rather than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis. As IF3 levels drop, expression from an AUU-infC-lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation of infC. The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein. In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41-43S is seen. Previous studies have shown that the L20 protein negatively controls its own gene expression. Reduction of the cellular concentration of L20 derepresses the expression of an rplT-lacZ gene fusion, thus confirming autogenous regulation by L20.

Genome-tagged mice (GTM): two sets of genome-wide congenic strains.

An important approach for understanding complex disease risk using the mouse is to map and ultimately identify the genes conferring risk. Genes contributing to complex traits can be mapped to chromosomal regions using genome scans of large mouse crosses. Congenic strains can then be developed to fine-map a trait and to ascertain the magnitude of the genotype effect in a chromosomal region. Congenic strains are constructed by repeated backcrossing to the background strain with selection at each generation for the presence of a donor chromosomal region, a time-consuming process. One approach to accelerate this process is to construct a library of congenic strains encompassing the entire genome of one strain on the background of the other. We have employed marker-assisted breeding to construct two sets of overlapping congenic strains, called genome-tagged mice (GTMs), that span the entire mouse genome. Both congenic GTM sets contain more than 60 mouse strains, each with on average a 23-cM introgressed segment (range 8 to 58 cM). C57BL/6J was utilized as a background strain for both GTM sets with either DBA/2J or CAST/Ei as the donor strain. The background and donor strains are genetically and phenotypically divergent. The genetic basis for the phenotypic strain differences can be rapidly mapped by simply screening the GTM strains. Furthermore, the phenotype differences can be fine-mapped by crossing appropriate congenic mice to the background strain, and complex gene interactions can be investigated using combinations of these congenics.