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.      

Stability and reactivation of tyrosine radicals from ribonucleotide reductase in tumor cells studied by ESR

Naturally occurring tyrosine radicals from the M2 subunit of ribonucleotide reductase (RR) have been recorded by ESR in proliferating ordinary Ehrlich-ascites (EA) tumor cells of mice. Tyrosine radicals are stable in EA cells at room temperature for 2 h. Up to 500 mW no microwave saturation occurs. The relatively high stability and non-saturation of tyrosine radicals in EA cells suggests a suitable protein conformation in the M2 subunit enabling a close contact between the tyrosine radical and the antiferromagnetic iron complex. This facilitates an ESR study of functionally essential tyrosine radicals of RR in EA cells at low temperature and recommends this cellular system for studying such processes as inhibition and activation, which change the content of tyrosine radicals of the proliferation-linked RR. Oxygen treatment of non-proliferating (quiescent) EA cells reactivates tyrosine radicals 2-3 fold as found in strongly proliferating cells. We conclude that in quiescent cells, suffering from a lack of oxygen due to their high density in the peritoneal cavity, a reactivation of tyrosine radicals occurs by oxidation of non-radical tyrosine residues of inactive M2 subunits.     

Social Learning of a Novel Foraging Skill by White-throated Magpie-jays (Calocitta formosa,Corvidae): a Field Experiment

The cooperatively breeding white-throated magpie-jay (Calocitta formosa) uses a variety of foraging tactics to find, harvest, and process food. Members of territorial groups forage together and may gain information about how to acquire food by observing each other. A field experiment was performed to determine whether a novel skill, door opening to gain access to food, was more rapidly acquired by members of groups in which a trained individual performed the skill. A higher proportion of jays in groups with behavioural ‘models’ (trained birds that opened doors in the presence of group members) acquired the door-opening skill than those groups without models. Young birds acquired the behaviour more frequently than older individuals. Aggressive behaviour at feeders may have affected the spread of the behaviour by reducing the likelihood that individuals performed the behaviour in the presence of other group members but may also have encouraged subordinate individuals to attempt door opening rather than ‘scrounge’.     

Characterization of the gene for the chromosomal dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990: the origin of the trimethoprim-resistant S1 DHFR from Staphylococcus aureus?

The gene for the chromosomally encoded dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990 has been cloned and characterized. The structural gene encodes a polypeptide of 161 amino acid residues with a calculated molecular weight of 18,417. This trimethoprim-sensitive (Tmps) DHFR, SeDHFR, differs in only three amino acids (Val-31-->Ile, Gly-43-->Ala, and Phe-98-->Tyr) from the trimethoprim-resistant (Tmpr) S1 DHFR encoded by transposon Tn4003. Since in addition the S. epidermidis gene also forms part of an operon with thyE and open reading frame 140 as in Tn4003, the chromosomally located gene encoding the Tmps SeDHFR is likely to be the molecular origin of the plasmid-located gene encoding the Tmpr S1 DHFR. Site-directed mutagenesis and kinetic analysis of the purified enzymes suggest that a single Phe-->Tyr change at position 98 is the major determinant of trimethoprim resistance.     

5-Formyl-2'-deoxyuridine: cytostatic and antiviral properties and possible modes of action.

5-Formyl-2'-deoxyuridine (fdUrd) was prepared by a new method starting from thymidine and investigated for its influence both on proliferation of cultured mammalian cells and virus replication in vitro. The compound was found to have strong cytostatic and antiviral properties: 50% inhibition of proliferation of BHK 21/C13 cells or Ehrlich ascites tumour cells (EAT) was obtained at 4 - 10(-6) and 6 - 10(-6) M, respectively, while the treatment of pseudorabies virus with the same concentration resulted in about 1.5 log reduction of virus yield. A concentration of 1 - 10(-4) M inhibited cell proliferation by 80 to 100% while the virus yield was reduced by more than 3 orders of magnitude. All inhibitions can be prevented by thymidine.--DNA synthesis of EAT cells in vitro, as estimated by incorporation of [32P]-phosphate or low concentrations of [3H]-thymidine, was inhibited. Further biochemical experiments have provided indirect evidence that the compound is phosphorylated by thymidine and thymidylate phosphorylating enzymes. An inhibition of cell free DNA synthesis was found to be depending on a given period of preincubation with the compound (supposed to be needed for the formation of fdUrd 5'-triphosphate). This suggests that the 5'-triphosphate of fdUrd is an inhibitor of DNA polymerases and--by analogy with experiments with 5-formyluridine-5'-triphosphate and RNA polymerases [14]--may be used as an affinity label for this group of enzymes. It is concluded that the described cytostatic and antiviral effects of fdUrd are due to an intracellular "lethal" synthesis of the relevant phosphates which inhibit thymidylate synthetase (as had been found earlier to occur with the chemically prepared nucleotide in cell free extracts [1, 2]) and DNA synthesizing enzymes.     

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence‐capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence‐capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence‐capture recovered 80%–90% of the control sample species. DNA sequence‐capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low‐cost, whereas DNA‐sequence capture for biodiversity assessment is still in its infancy, is more time‐consuming but provides more taxonomic assignments.