A Rapid Method for Preparing High Quality Alizarin Stained Skeletons of Adult Mice

A simple three-day technique is described for preparing completely cleared and high quality alizarin stained total skeletons of adult mice. Unfixed specimens are partially macerated during staining. Older specimens are heated for 15 min in 1% KOH. A heated solution of benzyl and ethyl alcohol, glycerin, and water is used for final clearing and hardening. This procedure requires about 10 min work per specimen and greatly simplifies preparation of stained and cleared skeletons of adult mice. Another technique, giving slightly better preparations, but requiring 11-14 days, is also described.     

Structure and function of the (A)BC excinuclease of Escherichia coli

(A)BC excinuclease is the enzymatic activity resulting from the mixture of E. coli UvrA, UvrB and UvrC proteins with damaged DNA. This is a functional definition as new evidence suggests that the three proteins never associate in a ternary complex. The UvrA subunit associates with the UvrB subunit in the form of an A2B1 complex which, guided by UvrA's affinity for damaged DNA binds to a lesion in DNA and delivers the UvrB subunit to the damaged site. The UvrB-damaged DNA complex is extremely stable (t1/2 congruent to 100 min). The UvrC subunit, which has no specific affinity for damaged DNA, recognizes the UvrB-DNA complex with high specificity and the protein complex consisting of UvrB and UvrC proteins makes two incisions, the 8th phosphodiester bond 5' and the 5th phosphodiester bond 3' to the damaged nucleotide. (A)BC excinuclease recognizes DNA damage ranging from AP sites and thymine glycols to pyrimidine dimers, and the adducts of psoralen, cisplatinum, mitomycin C, 4-nitroquinoline oxide and interstrand crosslinks.     

Lipoprotein(a) in women twins: heritability and relationship to apolipoprotein(a) phenotypes.

Lp(a) is a unique lipoprotein consisting of an LDL-like particle and a characteristic protein, apo(a). Increased levels of Lp(a) constitute a risk factor for coronary heart disease. Variation in the size of the apo(a) protein is a phenotype controlled by the apo(a) gene on chromosome 6 and is related to Lp(a) plasma levels. Based on 169 MZ and 125 DZ adult female twin pairs, this study's purpose was to estimate the proportion of the variation in Lp(a) levels that is due to genetic influences and to determine the extent to which the apo(a) locus explains this heritability. Lp(a) levels were significantly more similar in MZ twins than in DZ twins: mean co-twin differences were 3.9 +/- 5.7 mg/dl and 16.0 +/- 19.9 mg/dl (P less than .001), respectively. Intraclass correlations were .94 in MZ twins and .32 in DZ twins, resulting in a heritability estimate of .94 (P less than .001). Heritability was then calculated using only co-twins with the same apo(a) phenotype: the heritability estimate decreased to .45 but was still highly significant (P less than .001). Therefore, on the basis of heritability analysis of women twins, Lp(a) levels are almost entirely genetically controlled. Variation at the apo(a) locus contributes to this heritability, although other genetic factors could be involved.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Bloom Syndrome and Maternal Uniparental Disomy for Chromosome 15

Bloom syndrome (BS) is an autosomal recessive disorder characterized by increases in the frequency of sister-chromatid exchange and in the incidence of malignancy. Chromosome-transfer studies have shown the BS locus to map to chromosome 15q. This report describes a subject with features of both BS and Prader-Willi syndrome (PWS). Molecular analysis showed maternal uniparental disomy for chromosome 15. Meiotic recombination between the two disomic chromosomes 15 has resulted in heterodisomy for proximal 15q and isodisomy for distal 15q. In this individual BS is probably due to homozygosity for a gene that is telomeric to D15S95 (15q25), rather than to genetic imprinting, the mechanism responsible for the development of PWS. This report represents the first application of disomy analysis to the regional localization of a disease gene. This strategy promises to be useful in the genetic mapping of other uncommon autosomal recessive conditions.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.