Chiasma interference as a function of genetic distance.

For many organisms, meiotic double crossing over is less frequent than expected on the assumption that exchanges occur at random with respect to each other. This "interference," which can be almost total for nearby intervals, diminishes as the intervals in which the double crossovers are scored are moved farther apart. Most models for interference have assumed, at least implicitly, that the intensity of interference depends inversely on the physical distance separating the intervals. However, several observations suggest that interference depends on genetic distance (Morgans) rather than physical distance (base pairs or micrometers). Accordingly, we devise a model in which interference is related directly to genetic distance. Its central feature is that recombinational intermediates (C's) have two fates--they can be resolved with crossing over (Cx) or without (Co). We suppose that C's are distributed at random with respect to each other (no interference); interference results from constraints on the resolution of C's. The basic constraint is that each pair of neighboring Cx's must have between them a certain number of Co's. The required number of intervening Co's for a given organism or chromosome is estimated from the fraction of gene conversions that are unaccompanied by crossover of flanking markers. The predictions of the model are compared with data from Drosophila and Neurospora.

Topical reversion at the HIS1 locus of Saccharomyces cerevisiae. A tale of three mutants.

Mutants of the HIS1 locus of the yeast Saccharomyces cerevisiae are suitable reporters for spontaneous reversion events because most reversions are topical, that is, within the locus itself. Thirteen mutations of his1-1 now have been identified with respect to base sequence. Revertants of three mutants and their spontaneous reversion rates are presented: (1) a chain termination mutation (his1-208, née his1-1) that does not revert by mutations of tRNA loci and reverts only by intracodonic suppression; (2) a missense mutation (his1-798, née his1-7) that can revert by intragenic suppression by base substitutions of any sort, including a back mutation as well as one three-base deletion; and (3) a -1 frameshift mutation (his1-434, née his1-19) that only reverts topically by +1 back mutation, +1 intragenic suppression, or a -2 deletion. Often the +1 insertion is accompanied by base substitution events at one or both ends of a run of A's. Missense suppressors of his1-798 are either feeders or nonfeeders, and at four different locations within the locus, a single base substitution encoding an amino acid alteration will suffice to turn the nonfeeder phenotype into a feeder phenotype. Late-appearing revertants of his1-798 were found to be slowly growing leaky mutants rather than a manifestation of adaptive mutagenesis. Spontaneous revertants of his1-208 and his1-434 produced no late-arising colonies.

Is there a high rate of mitotic recombination between the loci encoding immunoglobulin VH and CH regions in gonial cells?

Rabbit sera from approximately 6000 offspring of matings informative for recombination at the immunoglobulin (Ig) heavy chain locus were tested with allotype antisera by double diffusion in gel. Seven recombinants were found, R1K-R7K, and in every case the recombinational event had taken place in the male parent. Two single males each fathered two recombinant offspring: R2K, R4K and R5K, R6K. Four further recombinants, reported from other laboratories as well as two new, as yet not fully documented recombinants in our laboratory, also occurred in the male parent. The recombinational events either separated the a locus which encodes the VH region of Ig, from the loci encoding its CH region or separated a-ms from the remaining linked loci d-e-f-g. The recombination frequency is 7/6142, approximately 0.1%. Our findings suggests that the recombinations took place in mitotic divisions during spermatogenesis. This work is the first evidence of gonial crossing-over in a mammal.

A model of bursal colonization.

In the chicken, and perhaps in all birds, the development of the cells which form antibodies, the B-cells, differs substantially from that in mammals. In birds, committed B-cells colonize a specialized organ, the bursa of Fabricius, which consists of some 10(4) follicles. Diversification, i.e., the development of the antibody repertoire, takes place in bursal follicles by a process termed "gene conversion." The avian bursa is easily accessible experimentally, and in the chicken, it has been the subject of extensive research. As an aid to experimentation in this field, we present here a formal mathematical model of bursal development. Formulae are derived which allow one to estimate the number and sizes of B-cell clones in bursal follicles, and hence the size of the overall antibody repertoire. Particular attention is paid to the problem of estimating experimental errors.

Looping out and deletion mechanism for the immunoglobulin heavy-chain class switch.

In the mouse pre-B-cell line 18-81, cells can switch production in vitro from immunoglobulin mu chain to gamma 2b chain. The gene encoding the gamma 2b chain is created by a rearrangement of the mu gene. This rearrangement always takes place within a homolog. In cells with a gamma 2b gene, most of the time the gene segment encoding the constant region of the mu chain is deleted, but often the rearrangement leads to cells that produce no immunoglobulin, and all DNA sequences are retained. The latter result is due to an inversion. Inversions exclude the unequal sister chromatid exchange model of the heavy-chain class switch. Looping out is an intermediate step in the process of generating an inversion. Our findings demonstrate that the switch rearrangement occurs by looping out and deletion.

Scope of action of the immunoglobulin mutator system.

The authors have developed a method to measure the rate of spontaneous mutations taking place in IgH, the gene encoding the immunoglobulin heavy chain. When an amber chain-termination codon mutates to a sense codon, translation of the polypeptide chain will be completed, and mutant cells producing the heavy chain can be detected with a fluorescent labelled antibody. The protocol used is the compartmentalization test which minimizes any effect of selection. In subclones of the pre-B lymphocyte line 18-81, the spontaneous mutation rate in the part of IgH encoding the variable region is somewhat greater than 10(-5) mutations per base pair per generation. This supports the hypothesis that hypermutation is not dependent on cell stimulation by an antigen. In a hybrid between a cell of this line and a myeloma (which represents the terminal stage of the B-cell lineage), the mutation rate was too low to be determined by this test, less than 10(-9). When the same loss to gain procedure system was used with an opal chain-terminating codon in the part of IgH encoding the constant region (C mu), a high rate of reversion by deletion was found. Long (more than one exon) and short (less than one exon) deletions occurred at rates of 1.7 x 10(-5) and 1.4 x 10(-7) per generation, respectively. It is thought that the high rate of deletion is not related to somatic hypermutation but rather to DNA rearrangement during the heavy-chain class switch, which is occurring in these pre-B cell lines. The point mutation rate was too low to be detected above the background of deletion mutants, less than 5 x 10(-8). The immunoglobulin mutator system works weakly, if at all, on two other, nonimmunoglobulin, genes tested: B2m (beta 2 microglobulin) and the gene for ouabain resistance.

Effects of sibutramine plus orlistat in obese women following 1 year of treatment by sibutramine alone: a placebo-controlled trial.

OBJECTIVE: This study assessed whether adding orlistat to sibutramine would induce further weight loss in patients who previously had lost weight while taking sibutramine alone. RESEARCH METHODS AND PROCEDURES: Patients were 34 women with a mean age of 44.1 +/- 10.4 years, weight of 89.4 +/- 13.8 kg, and body mass index (BMI) of 33.9 +/- 4.9 kg/m2 who had lost an average of 11.6 +/- 9.2% of initial weight during the prior 1 year of treatment by sibutramine combined with lifestyle modification. Patients were randomly assigned, in double-blind fashion, to sibutramine plus orlistat or sibutramine plus placebo. In addition to medication, participants were provided five brief lifestyle modification visits during the 16-week continuation trial. RESULTS: Mean body weight did not change significantly in either treatment condition during the 16 weeks. The addition of orlistat to sibutramine did not induce further weight loss as compared with treatment by sibutramine alone (mean changes = +0.1 +/- 4.1 kg vs. +0.5 +/- 2.1 kg, respectively). DISCUSSION: These results must be interpreted with caution because of the study's small sample size. The findings, however, suggest that the combination of sibutramine and orlistat is unlikely to have additive effects that will yield mean losses > or =15% of initial weight, as desired by many obese individuals.

Quantitative determination of high-density lipoprotein cholesterol by agarose gel electrophoresis.

We have developed a procedure for the determination of high-density lipoprotein cholesterol by agarose gel electrophoresis. Only 2 micro L of sample was applied to the gel. After electrophoresis at 90 V for 35 min, an enzymatic cholesterol reagent was applied. After a 30-min incubation, the high-density lipoprotein cholesterol was quantified by densitometry. Precision for this measurment approaches that reported for the heparin-manganese/Abell-Kendall method (Clin. Chem, 25: 596--609, 1979). We evaluated accuracy by comparing high-density lipoprotein cholesterol concentration measured by electrophoresis to that determined in the Framingham Heart Study procedure (J. Biol. Chem. 195: 357, 1952). The resulting correlation was excellent. By the paired Student's t-test, there was no significant difference between the two methods. The proposed method gives a linear standard curve when the concentration of total cholesterol is between 1.0 and 3.5 g/L. By accurate quantitation of high-density lipoprotein cholesterol, agarose gel electrophoresis can aid in assessment of coronary heart disease risk for a large segment of the population.

Gene 24-controlled osmotic shock resistance in bacteriophage T4: probable multiple gene functions.

By use of mixed infections with conditional lethal mutations in the head genes and an osmotic shock-resistant mutant we have demonstrated that osmotic shock resistance is controlled by gene 24. Using acrylamide gel electrophoresis combined with the "immune replicate" technique, we confirmed the positions of gene products 24 and 24* (P24 and P24*). In this paper we have still used the notation "P24," etc., for designating the product of gene 23, etc., although we prefer and use in general the designation "gp23" as introduced by Casjens and King (Annu. Rev. Biochem. 44:585, 1975). The reason for using the old notation is because the illustrations were prepared several years ago.) P24 ts showed a significantly slower mobility. Both osmotic shock-resistant and -sensitive mature phages contain 24*. Giants constructed with the Osr phage showed the same surface lattice as normal phage. Through temperature-shift experiments with 24(tsL90) alone and in combinations, we studied the phages which are matured after the shift to permissive temperature in the absence of new protein synthesis. Our results strongly suggest that only a fraction of the total phage complement of gene 24-controlled proteins is involved in determining the phenotype of shock resistance, and the remainder is necessary to mature the head.

Measurements of mutation rates in B lymphocytes.

It is established that somatic mutation is an important source of antibody diversity in vivo. It is also established that Igh-V gene segments are hypermutable in vitro. This is not a completely satisfactory situation. While there is no reason to believe that Igh-V genes are not hypermutable in vivo as well, direct experimental evidence is lacking. Perhaps experiments with transgenic mice will soon fill this gap. It is not so clear how much higher than normal the rate of hypermutation is. As far as we are aware, there are no direct measurements of mutation rates per base pair per cell generation in mammals, certainly not for lymphocyte cell lines. For a variety of reasons, it is difficult to measure very low mutation rates. The general consensus is that the normal rate should be somewhere between 10(-10) and 10(-12) mutations per base pair per cell generation. Therefore, an experiment designed to directly determine a rate using the compartmentalization test would involve hundreds of cultures, each containing at least 10(9) cells. It is not a trivial problem to find one or a few mutants among so many cells. It is simple to study mutation to resistance to a drug, for example, ouabain or azaguanine, but, as we discussed, there are technical and conceptual pitfalls. The vast excess of dead cells influences the growth of a few mutant cells, particularly in lymphocyte cell lines. Even if this problem could be solved, the mutation rate so obtained would be "per gene(s)" and not "per base pair". The problems associated with cytotoxic agents can be avoided by immunofluorescence methods in conjunction with selective cloning or cell sorting. Using these techniques, we have carried out extensive experiments to determine whether the immunoglobulin mutator system acts, at least partially, on genetic elements other than those in or near the heavy chain variable region gene segment. For an opal termination codon in a heavy chain constant region gene segment, the rate of reversion was less than 10(-7) per base pair per cell generation. This upper limit was fixed by the high rate of small deletions at the heavy chain locus. For an allotype mutation at B2m, the gene encoding beta 2 microglobulin, the rate of mutation was less than 10(-8). This upper limit could be lowered by at least two orders of magnitude by using a high-speed cell sorter.