A method for generating selective DNA probes for the analysis of C-negative regions in human chromosomes

Linker-adapter polymerase chain reaction (LA-PCR) is among the most efficient techniques for whole genome DNA amplification. The key stage in LA-PCR is the hydrolysis of a DNA sample with restriction endonucleases, and the choice of a restriction endonuclease (or several endonucleases) determines the composition of DNA probes generated in LA-PCR. Computer analysis of the localization of the restriction sites in human genome has allowed us to propose an efficient technique for generating DNA probes by LA-PCR using the restriction endonucleases HaeIII and RsaI. In silico hydrolysis of human genomic DNA with endonucleases HaeIII and RsaI demonstrate that 100- to 1,000-bp DNA fragments are more abundant in the gene-rich regions. Applying in situ hybridization to metaphase chromosomes, we demonstrated that the produced DNA probes predominantly hybridized to the C-negative chromosomal regions, whereas the FISH signal was almost absent in the C-positive regions. The described protocol for generating DNA probes may be successfully used in subsequent cytogenetic analysis of the C-negative chromosomal regions.     

Behavior of hobo and P transposons in yellow2-717 unstable line of Drosophila melanogaster and its derivatives after crossing with a laboratory strain

Using fluorescent in situ hybridization technique (FISH), the frequency of hobo and P mobile elements transpositions on X chromosomes from the y2-717, isolated from the Uman' population of Drosophila melanogaster, as well as from its phenotypically normal and mutant derivatives, obtained as a result of crosses the males examined with the C(I)DX, ywf/Y females, was evaluated. It was demonstrated that the maximum frequency of hobo transpositions on X chromosomes of the males from derivative strains, subjected to repeated hobo-dysgenic crosses reached a value of 1.2 x 10(-2) per site per X chromosome per generation. The number of hobo copies in male X chromosomes from derivative strains was 3 times higher than in the original initial strain. Furthermore, the "old" hobo sites remained unchanged. In derivative strains, the frequency of hobo insertions was higher than that of excisions. One of the derivative strains, y1t-717alk3-2, was characterized by high intra-strain instability of hobo element localization. In the y2-717a1k3 and y1t-717alk3-2 strains a large inversion, In(1)1B; 13CD, was described. At the absence of the full-sized P element in the strains involved in crosses, maximum frequency of P element transpositions in the derivative strains reached a value of 1.2 x 10(-2) per site per X chromosome per generation.     

Multiple Anaphase-promoting Complex/Cyclosome Degrons Mediate the Degradation of Human Sgo1

Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion in early mitosis and, thus, prevents premature sister-chromatid separation. The protein level of Sgo1 is regulated during the cell cycle; it peaks in mitosis and is down-regulated in G₁/S. Here we show that Sgo1 is degraded during the exit from mitosis, and its degradation depends on the anaphase-promoting complex/cyclosome (APC/C). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Sgo1 in human cells. Sgo1 is ubiquitinated by APC/C bound to Cdh1 (APC/C^Cdh1) in vitro. We have further identified two functional degradation motifs in Sgo1; that is, a KEN (Lys-Glu-Asn) box and a destruction box (D box). Although removal of either motif is not sufficient to stabilize Sgo1, Sgo1 with both KEN box and D box deleted is stable in cells. Surprisingly, mitosis progresses normally in the presence of non-degradable Sgo1, indicating that degradation of Sgo1 is not required for sister-chromatid separation or mitotic exit. Finally, we show that the spindle checkpoint kinase Bub1 contributes to the maintenance of Sgo1 steady-state protein levels in an APC/C-independent mechanism.Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs in two steps in vertebrate cells (1-3). In prophase, cohesin is phosphorylated by mitotic kinases including Plk1 and removed from chromosome arms (1, 4). Then, cleavage of centromeric cohesin by separase takes place at the metaphase-to-anaphase transition to allow sister-chromatid separation (5). The shugoshin (Sgo) family of proteins plays an important role in the protection of centromeric cohesion (6, 7). Human cells depleted of Sgo1 by RNAi undergo massive chromosome missegregation (8-11). In cells with compromised Sgo1 function, centromeric cohesin is improperly phosphorylated and removed (4, 11), resulting in premature sister-chromatid separation. It has been shown recently that Sgo1 collaborates with PP2A to counteract the action of Plk1 and other mitotic kinases and to protect centromeric cohesin from premature removal (12-14). In addition, Sgo1 has also been shown to promote stable kinetochore-microtubule attachment and sense tension across sister kinetochores (8, 15). Thus, Sgo1 is crucial for mitotic progression and chromosome segregation.Orderly progression through mitosis is regulated by the anaphase-promoting complex/cyclosome (APC/C),² a large multiprotein ubiquitin ligase that targets key mitotic regulators for destruction by the proteasome (16). APC/C selects substrates for ubiquitination by using the Cdc20 or Cdh1 activator proteins to recognize specific sequences called APC/C degrons within target proteins (17). Several APC/C degrons have been characterized, including the destruction box (D box) and the Lys-Glu-Asn box (KEN box) (18, 19). The D box, with the consensus amino acid sequence of RXXLXXXN(X indicates any amino acid), are found in many APC/C substrates, including mitotic cyclins and are essential for their ubiquitin-mediated destruction. The KEN box, which contains a consensus KEN motif, is also found in several APC/C substrates and is preferentially but not exclusively recognized by APC/C^Cdh1. When APC/C is active, it directs progression through and exit from mitosis by catalyzing the ubiquitination and timely destruction of mitotic regulators, including cyclin A, cyclin B, and the separase inhibitor securin (16). The APC/C activity needs to be tightly controlled to prevent unscheduled substrate degradation. An important mechanism for APC/C regulation is the spindle checkpoint, which prevents the activation of APC/C and destruction of its substrates in response to kinetochores that have not properly attached to the mitotic spindle (20).Recent evidence shows that Sgo1 is a substrate of APC/C, and its protein levels oscillate during the cell cycle (8, 9). In this article we study the degradation of Sgo1 in human cells. We show that Sgo1 is degraded during mitotic exit, and this degradation depends on APC/C^Cdh1. We further show that both KEN and D boxes are required for Sgo1 degradation in vivo and ubiquitination in vitro. Removal of these motifs stabilizes Sgo1 in vivo. The prolonged presence of stable Sgo1 protein in human cells does not change the kinetics of chromosome segregation and mitotic exit. Therefore, a timely scheduled degradation of Sgo1 takes place but is not required for mitotic exit. Finally, we show that Bub1 regulates Sgo1 protein levels through a mechanism that does not involve APC/C-mediated degradation.     

Recombination map of the common shrew, Sorex araneus (Eulipotyphla, Mammalia)

The Eurasian common shrew (Sorex araneus L.) is characterized by spectacular chromosomal variation, both autosomal variation of the Robertsonian type and an XX/XY(1)Y(2) system of sex determination. It is an important mammalian model of chromosomal and genome evolution as it is one of the few species with a complete genome sequence. Here we generate a high-precision cytological recombination map for the species, the third such map produced in mammals, following those for humans and house mice. We prepared synaptonemal complex (SC) spreads of meiotic chromosomes from 638 spermatocytes of 22 males of nine different Robertsonian karyotypes, identifying each autosome arm by differential DAPI staining. Altogether we mapped 13,983 recombination sites along 7095 individual autosomes, using immunolocalization of MLH1, a mismatch repair protein marking recombination sites. We estimated the total recombination length of the shrew genome as 1145 cM. The majority of bivalents showed a high recombination frequency near the telomeres and a low frequency near the centromeres. The distances between MLH1 foci were consistent with crossover interference both within chromosome arms and across the centromere in metacentric bivalents. The pattern of recombination along a chromosome arm was a function of its length, interference, and centromere and telomere effects. The specific DNA sequence must also be important because chromosome arms of the same length differed substantially in their recombination pattern. These features of recombination show great similarity with humans and mice and suggest generality among mammals. However, contrary to a widespread perception, the metacentric bivalent tu usually lacked an MLH1 focus on one of its chromosome arms, arguing against a minimum requirement of one chiasma per chromosome arm for correct segregation. With regard to autosomal chromosomal variation, the chromosomes showing Robertsonian polymorphism display MLH1 foci that become increasingly distal when comparing acrocentric homozygotes, heterozygotes, and metacentric homozygotes. Within the sex trivalent XY(1)Y(2), the autosomal part of the complex behaves similarly to other autosomes.     

The structure of a conserved region of porcine genome, represented in human genome by chromosome 17

Radiation mapping of nine genes (H3F3B, HLR1, MYL4, STAT5B, THRA1, TOP2A, MCP1, NF1, and MPO) to porcine chromosome 12 was carried out. Also, subchromosomal location of the NF1 gene along with the two loci containing the DNA sequences homologous to the DNA of the two human BAC clones was determined. The NF1 position was ascertained via microdissection of chromosome 12 with subsequent PCR amplification of the gene fragment with specific primers. BAC clones were mapped using FISH. Comparative analysis of the gene order in porcine chromosome 12 and in the homologous human chromosome 17 was performed. It was demonstrated that the gene orders in these chromosomes differed relative to the position of the MPO gene.     

Genetic mechanisms of resistance and susceptibility to leukemia in Ayrshire and black pied cattle breeds determined by allelic distribution of gene Bola-DRB3

In the herds of Ayrshire and Black Pied cattle breeds of Russian selection, comparative analysis of allelic distribution of BoLA-DRB3 was performed in animal groups with different status of persistent lymphocytosis (PL) caused by the bovine leukemia virus (BLV). Alleles were typed by PCR-RFLP. Different spectra of BoLA-DRB3 alleles mediating susceptibility and resistance to leukemia were detected in the studied breeds. The role of amino acid motives in beta 1 domain of BoLA-DRB3 antigens was confirmed: ER (in positions 70-71), in resistance to leukemia and VDTY and VDTV (75-78), in susceptibility to leukemia. The nucleotide sequence of allele BoLA-DRB3.2*7 with deletion of codon 65, which resulted in the changed conformation of the corresponding antigen molecule, was associated with resistance to PL. Cows of Black Pied and Ayrshire breeds with genotypes coding VDTY/VDTV (RR = 11.67, P = 0.014) and VDTY/VDTY (RR = 4.71, P = 0.022), respectively, were shown to be susceptible to PL. The role of heterozygosity level was demonstrated (estimated by BoLA-DRB3 alleles and by amino acid motives in positions 75-78 of the antigen) as an unspecific factor of resistance to PL. The lowest heterozygosity level by amino acid motives (75-78) was revealed in PL animals, for which sample inbreeding coefficients were detected: F = 0.324 and F = 0.084 in Ayrshire and Black Pied breeds, respectively.     

Comparative analysis of Ayrshire and Black Pied cattle breeds by histocompatibility markers

Distribution of BoLA-A antigens and BoLA-DRB3 alleles was studied by means of the microlymphocytotoxic test (BoLA-A) and the PCR-RFLP method (BoLA-DRB3) using restriction endonucleases RSAI, HaeIII, and XhoII in Ayrshire (n = 127) and Black Pied (n = 129) cattle breeds. Comparative analysis of profiles for class I antigens revealed significant differences in the frequencies of antigens W2, W6, W10, W31, W44, W15, and W19 (P > 99%). The studied breeds also differ in the spectrum of BoLA-DRB3 alleles and distribution of their frequencies. Heterogeneous allele frequency profile was detected in Ayrshire cattle: five of 18 detected alleles (DRB3.2*7, *8, *10, *24, and *28) accounted for 77%. Allele DRB3.2*7 (37.6%), which is classed with rare alleles in Black Pied cattle is the most common in Ayrshire cattle. The observed heterozygosity level in the combined sample of Black Pied breed (0.836) is higher than in Ayrshire breed (0.070). In both breeds, the heterozygosity level was studied in the groups of healthy and ill with persistent lymphocytosis (caused by bovine leukemia virus) animals and in the group of virus carriers in Ayrshire breed. In ill animals, a decrease in the observed heterozygosity level was detected, as compared to healthy animals and the expected heterozygosity level. The observed heterozygosity level exceeds the expected one in virus carriers. The detected features of the heterozygosity level in the studied groups allow the heterozygosity level for locus BoLA-DRB3 to be considered a nonspecific factor of resistance to leukemia and are heterozygous animals to have higher resistance to bovine leukemia. The presence of a larger proportion of highly productive animals (the annual productivity of more than 7000 kg) in the group of ill Ayrshire cattle animals, as compared to healthy animals to established. To increase resistance to bovine leukemia, the obtained data indicate the importance of the control of heterozygosity level and genetic diversity for gene BoLA-DRB3 in cattle herds.     

Interaction of mutant alkaline phosphatase precursors with membrane phospholipids in vivo and in vitro.

Positively charged amino acid residues in the N-terminal domain of the signal peptides of secreted proteins are thought to interact with negatively charged anionic phospholipids during the initiation of secretion. To test this hypothesis, substitutions of the uncharged Ala or the negatively charged Glu residue for the positively charged Lys-20 of the N-terminus of the signal peptide of Escherichia coli alkaline phosphatase were introduced using a modified method of oligonucleotide-directed mutagenesis. We found that Lys-20 is involved in the interaction of the signal peptide with anionic phospholipids in vivo and effects the efficiency of insertion of the signal peptide of isolated precursor into model phospholipid membranes in vitro. We also show that the efficiency of signal peptide insertion into the lipid bilayer depends on the fluidity of the bilayer.     

Graded requirement for the spliceosome in cell cycle progression

Genome stability is ensured by multiple surveillance mechanisms that monitor the duplication, segregation, and integrity of the genome throughout the cell cycle. Depletion of components of the spliceosome, a macromolecular machine essential for mRNA maturation and gene expression, has been associated with increased DNA damage and cell cycle defects. However, the specific role for the spliceosome in these processes has remained elusive, as different cell cycle defects have been reported depending on the specific spliceosome subunit depleted. Through a detailed cell cycle analysis after spliceosome depletion, we demonstrate that the spliceosome is required for progression through multiple phases of the cell cycle. Strikingly, the specific cell cycle phenotype observed after spliceosome depletion correlates with the extent of depletion. Partial depletion of a core spliceosome component results in defects at later stages of the cell cycle (G2 and mitosis), whereas a more complete depletion of the same component elicits an early cell cycle arrest in G1. We propose a quantitative model in which different functional dosages of the spliceosome are required for different cell cycle transitions.