The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Callus induction and shoot regeneration in Sempervivum tectorum

Leaf and shoot explants of Sempervivum tectorum L., taken from 14- and 30-day-old plants germinated in vitro, have been studied by using Murashige-Skoog and White basal media with cytokinins (benzyladenine, kinetin) and auxins (indoleacetic acid, naphthaleneacetic acid, indolebutyric acid) in various concentrations. Explants taken from 14-day-old plants died but 30-day-old leaves and shoots produced yellow and soft, as well as green and hard calluses on Murashige-Skoog medium with 4.4–8.8 M benzyladenine and 0.57 M indoleacetic acid. Shoot organogenesis was induced from green, hard callus in a medium with 2.2 M benzyladenine plus either 1.1 M indoleacetic acid or 2.5 M indolebutyric acid. Whole plants were grown on Murashige-Skoog medium without plant growth regulators. On the other hand, White medium was not suitable for raising Sempervivum tectorum in vitro.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - MS Murashige-Skoog - NAA napthaleneacetic acid - W White     

Flow cytometry reveals different lag times in rapid cytoplasmic calcium elevations in human neutrophils in response to N-formyl peptide

Flow cytometric analyses were performed to study intracellular single-cell calcium transients ([Ca²⁺]_i) in suspended human neutrophils during the initial phase of N-formyl peptide stimulation. Thereby, two neutrophil populations became apparent. Early maximally Ca²⁺-responding (high fluorescence) neutrophils and not-yet Ca²⁺-responding (low fluorescence) neutrophils, but no neutrophils with intermediate levels of [Ca²⁺]_i, were detected. Within 7 s the number of low fluorescence neutrophils decreased and the number of high fluorescence neutrophils increased maximally. This suggests that [Ca²⁺]_i transients occurred abruptly in individual neutrophils within a time interval below 1 s. At lower N-formyl peptide concentrations the lag times of individual neutrophils and the interval time of maximal activation of the [Ca²⁺]_i-responding neutrophil population increased, however the percentage of [Ca²⁺]_i-responding cells decreased. Surprisingly, no influence of the N-formyl peptide concentration on the [Ca²⁺]_i-induced fluorescence signal of the individual cell was observed: it was always in an almost maximal range or not responding. In parallel, binding studies performed with fluorescein-labeled N-formyl peptide revealed that the heterogeneity of [Ca²⁺]_i-responding cells cannot be explained by different receptor occupancy. In summary, this study demonstrates that [Ca²⁺]_i transients induced by N-formyl peptides in suspended individual human neutrophils occur very rapidly in an almost “all-or-none manner” and that the mean increasing fluorescence signal of a calcium indicator within a whole neutrophil population results from varying lag times of the individual cells, rather than from the mean simultaneous progress of many cells. © 1993 Wiley-Liss, Inc.     

Biophysical studies of infectious pancreatic necrosis virus.

The molecular weight of infectious pancreatic necrosis virus (IPNV) has been determined by analytical ultracentrifugation and dynamic light scattering. The sedimentation coefficient of the virus was found to be 435S. The average value for molecular weight is (55 +/- 7) x 106. The virus genome consists of two segments of double-stranded RNA (molecular weights, 2.5 x 106 and 2.3 x 106), which represents 8.7% of the virion mass. The capsid protein moiety of IPNV consists of four species of polypeptides, as determined by polyacrylamide gel electrophoresis. The number of molecules of each polypeptide in the virion has been determined. There are 22 molecules of the internal polypeptide alpha (molecular weight, 90,000), 544 molecules of the outer capsid polypeptide beta (molecular weight, 57,000), and 550 and 122 molecules, respectively, of the internal polypeptides gamma1 (molecular weight, 29,000) and gamma2 (molecular weight, 27,000). IPNV top component contains only the beta polypeptide species, and its molecular weight is estimated to be 31 x 106. The hydrodynamic diameter and electron microscopic diameter (calculated by catalase crystal-calibrated electron microscopy) of IPNV was compared with those of reovirus and encephalomyocarditis virus. Due to the swelling of the outer capsid, reovirus particles were found to be much larger when hydrated (96-nm diameter) than when dehydrated (76-nm diameter), having a large water content content and low average density. In contrast, IPNV particles are more rigid, having nearly the same average diameter under hydrous (64 nm) as under anhydrous conditions (59.3 nm). Encephalomyocarditis virus has a very low water content and does not shrink at all when prepared for electron microscopy.     

Determination of the basicity of Mannich ketones by capillary electrophoresis

Capillary electrophoretic (CE) method to characterise Mannich ketones (MKs) containing morpholine moiety was developed. Basicity (pKa,exp) of the MKs was characterised by measured data derived from the electrophoretic mobility values obtained in the CE separation. The MKs were found to be weaker bases than the parent morpholine molecule itself and the experimentally determined basicity values proved to be dependent on the chemical structure of the MKs. Since the basicity of the MKs has an influence on their reactivity and biological activity it seems to be useful to determine experimentally the pKa,exp values of the newly synthesised compounds to support rational drug design or screening of the molecule libraries.     

Continued proteomic analysis of Mycobacterium leprae subcellular fractions

Recently the sequence of the Mycobacterium leprae chromosome, the only known obligate intracellular mycobacterium, was completed. It has a dramatic reduction in functional genes, with a coding capacity of only 49.5%, the lowest one so far observed among bacterial genomes. The leprosy bacillus seems to preserve a minimal set of genes that allows its survival in the host. The identification of genes that are actually expressed by the bacterium is of high significance in the context of mycobacterial pathogenesis. In this current study, a proteomic approach was undertaken to identify the proteins present in the soluble/cytosol and membrane subcellular fractions obtained from armadillo derived M. leprae. Proteins from each fraction were separated by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry. A total of 147 protein spots were identified from 2-DE patterns and shown to comprise products of 44 different genes, twenty eight of them corresponding to new proteins. Additionally, two highly basic proteins (with pI >10.0) were isolated by heparin affinity chromatography and identified by N-terminal sequencing. This study constitutes the first application of proteomics to a host-derived Mycobacterium.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.