Topologically constrained domains of supercoiled DNA in eukaryotic cells

The size of supercoiled, topologically constrained DNA domains within the squamous carcinoma cell line SQ-20B were determined by direct comparison with a panel of irradiated supercoiled plasmid DNAs. Loss of supercoiling in plasmids was determined by gel electrophoresis and in cells by nucleoid flow cytometry. Comparison of dose-response data for plasmid relaxation with that obtained from SQ-20B cells enabled a direct estimation of supercoil target size in these cells. Plasmids pUCD9P (3.9 kbp), pXT-1 (10.1 kbp), pdBPV-MMT-neo (14.6 kbp), pRK290 (20.0 kbp), and R6K (38 kbp) were used and analyzed under the same exposure conditions as nucleoid DNA. Two sizes of topologically closed domains were found in nucleoids of 0.51+/-0.17Mbp and 1.34+/-0.3 Mbp. In an attempt to relate these large-scale organizations of DNA with function, cells were exposed to the DNA topoisomerase II inhibitor, VP16 and the G1/S cell cycle blocking agent mimosine. A 1 h exposure to VP16 was effective in reducing DNA synthesis which was associated with a parallel increase in nucleoid supercoiling. Addition of the G1 > S inhibitor mimosine enhanced both responses. It is concluded that chromosomes and interphase nuclei are organized into at least two sizes of topologically constrained domains of DNA which may have functional relevance to the control and execution of DNA synthesis.     

Antibody VH domains as small recognition units

To develop immunoglobulin based recognition units of minimum size, a human heavy chain variable domain (VH) was designed for selection of phage displayed VH. Non-specific binding of the VH through its interface for the light chain variable domain (VL) was prevented through three mutations (G44E, L45R and W47G) in this interface. These mutations were introduced to mimic camelid antibody heavy chains naturally devoid of light chain partners. The third hypervariable loop of the modified VH was then randomised to yield a repertoire of 2 x 10(8) independent clones, which was displayed on phage and selected through antigen binding. VH clones specific for hapten and protein antigens were isolated. Soluble VH was expressed with an isoleucine residue at position 47 to improve expression and stability compared to VH containing a glycine residue at this position, which however was preferable for phage selection. Affinities of soluble VH for hapten were between 100 nM and 400 nM. The VH domains were highly specific, stable and well expressed in Escherichia coli. These positive biophysical properties and their small size make them attractive for biotechnological applications.     

Stretched polytene chromosomes--model for studying the functional organization of eukaryotic chromosomes

To localize functional loci on cytological maps of polytene chromosomes we propose to use 10-100 times stretched chromosomes. Three different ways of stretchening are briefly considered: the squash tissue preparation, when chromosomes are stretched by hydrodynamical forces; the treatment of isolated polytene chromosomes in 10-minus 4M EDTA OR 0.8M NaCL with subsequent change of these solution for saline when abrupt structural changes occur in chromosomes and they become morphologically homogeneous threads (Gruzdev and Belaya, 1973); and, finally, the use of microneedles of the micromanipulator. After an intense (ca. 100 times) stretchening, the autoradiography is sufficient to localize the loci within one micron length of double helical DNA molecule.     

MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains

The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins.     

Phosphorylation of serine residues in the N-terminal domains of eukaryotic type I topoisomerases

OBJECTIVE: Some countries have regulations against using a cellular telephone while driving. We used ecologic analysis to evaluate cellular telephone use and motor vehicle collisions in a city without such regulations. METHODS: We studied locations in Toronto, Ontario (n = 75) that were hazardous (total collisions = 3,234) and tested whether increases in collision rates from 1984 to 1993 correlated with increases in telephone usage over the same time interval. RESULTS: Locations with the largest increases in collision rates tended to have the smallest increases in estimated cellular telephone usage. Yet extreme assumptions about potential protective effects from cellular telephones failed to explain the magnitude observed. CONCLUSIONS: The effects of cellular telephones on driving ability are small relative to the biases in ecologic analysis. Claims from industry, which argue that cellular telephones are not dangerous based on ecologic analysis, can be misleading in the policy debate about whether to regulate cellular telephone use while driving.     

Expression of var genes located within polymorphic subtelomeric domains of Plasmodium falciparum chromosomes

Plasmodium falciparum var genes encode a diverse family of proteins, located on the surfaces of infected erythrocytes, which are implicated in the pathology of human malaria through antigenic variation and adhesion of infected erythrocytes to the microvasculature. We have constructed a complete representative telomere-to-telomere yeast artificial chromosome (YAC) contig map of the P. falciparum chromosome 8 for studies on the chromosomal organization, distribution, and expression of var genes. Three var gene loci were identified on chromosome 8, two of which map close to the telomeres at either end of the chromosome. Analysis of the previously described chromosome 2 contig map and random P. falciparum telomeric YAC clones revealed that most, if not all, 14 P. falciparum chromosomes contain var genes in a subtelomeric location. Mapping the chromosomal location of var genes expressed in a long-term culture of the P. falciparum isolate Dd2 revealed that four of the five different expressed var genes identified map within subtelomeric locations. Expression of var genes from a chromosomal domain known for frequent rearrangements has important implications for the mechanism of var gene switching and the generation of novel antigenic and adhesive phenotypes.     

Structural maintenance of chromosomes protein C-terminal domains bind preferentially to DNA with secondary structure

Structural maintenance of chromosomes (SMC) proteins interact with DNA in chromosome condensation, sister chromatid cohesion, DNA recombination, and gene dosage compensation. How individual SMC proteins and their functional domains bind DNA has not been described. We demonstrate the ability of the C-terminal domains of Saccharomyces cerevisiae SMC1 and SMC2 proteins, representing two major subfamilies with different functions, to bind DNA in an ATP-independent manner. Three levels of DNA binding specificity were observed: 1) a >100-fold preference for double-stranded versus single-stranded DNA; 2) a high affinity for DNA fragments able to form secondary structures and for synthetic cruciform DNA molecules; and 3) a strong preference for AT-rich DNA fragments of particular types. These include fragments from the scaffold-associated regions, and an alternating poly(dA-dT)-poly(dT-dA) synthetic polymer, as opposed to a variety of other polymers. Reannealing of complementary DNA strands is also promoted primarily by the C-terminal domains. Consistent with their in vitro DNA binding activity, we show that overexpression of the SMC C termini increases plasmid loss without altering viability or cell cycle progression.     

Poly(A) polymerase contains multiple functional domains

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS.     

An eukaryotic RuvB-like protein (RUVBL1) essential for growth

A human protein (RUVBL1), consisting of 456 amino acids (50 kDa) and highly homologous to RuvB, was identified by using the 14-kDa subunit of replication protein A (hsRPA3) as bait in a yeast two-hybrid system. RuvB is a bacterial protein involved in genetic recombination that bears structural similarity to subunits of the RF-C clamp loader family of proteins. Fluorescence in situ hybridization analysis demonstrated that the RUVBL1 gene is located at 3q21, a region with frequent rearrangements in different types of leukemia and solid tumors. RUVBL1 co-immunoprecipitated with at least three other unidentified cellular proteins and was detected in the RNA polymerase II holoenzyme complex purified over multiple chromatographic steps. In addition, two yeast homologs, scRUVBL1 and scRUVBL2 with 70 and 42% identity to RUVBL1, respectively, were revealed by screening the complete Saccharomyces cerevisiae genome sequence. Yeast with a null mutation in scRUVBL1 was nonviable. Thus RUVBL1 is an eukaryotic member of the RuvB/clamp loader family of structurally related proteins from bacteria and eukaryotes that is essential for viability of yeast.     

Maternal origin of inv dup(15) chromosomes in infantile autism

Six male patients with infantile autism and an extra inverted duplicated chromosome 15[inv dup(15)] were reported in a previous study. These patients had four copies of the chromosome region 15pter-q13, or an inv dup(15)(pter-->q13; q13-->pter). In this new study, DNA from the families of four of the patients were analysed using Southern based RFLPs and microsatellite polymorphisms from the region. In all four cases the inv dup(15) chromosome was of maternal origin. Furthermore, the data suggests that it originated in the maternal meiotic process rather than in an early mitosis in the developmental process of the embryo. The extra chromosome contained material from both of the maternally derived 15-chromosomes. Based on the molecular data presented here, a model for the origin of chromosome markers of this type is proposed.     

（Co78Fe22）3V中的层错和反相畴界

比较了淬火无序态（Ｃｏ７８Ｆｅ２２）３Ｖ在经过冷加工后退火和未经冷加工直接退火两种情况下的显微组织，发现在无序一有序转变过程中出现的晶体缺陷和有序态下（再结晶晶粒内）出现的是体缺陷不同，前者主要有广泛的反相畴界网络，后者主要是大量的内禀层错和孪晶，实验结果表明，有序态下超点阵内禀层错（ＳＩＳＦ）能比反相畴界（ＡＰＢ）能低得多，无序一有序转变过程中出现的位移矢量为１／６〈１１２，层错（ＳＦ）通过和Ａ     

Reduction of hospital stay in surgical units (author's transl)

Reduction of hospital stay increases the surgical efficiency and reduces the danger of bacterial hospitalism. It is to be accomplished by: 1. admittance of patients with regard to the operative capacity; 2. out-patient diagnosis and preoperative anaesthesiologie care; 3. early postoperative discharge; 4. reduction of septic surgery.     

Geometric invariant core for the V(L) and V(H) domains of immunoglobulin molecules

A new algorithmic method for identifying a geometric invariant of protein structures, termed geometrical core, is developed. The method used the matrix of C(alpha)-C(alpha) distances and does not require the usual superposition of structures. The result of applying the algorithm to 53 immunoglobulin structures led to the identification of two geometrical core sets of C(alpha) atoms positions for the V(L) and V(H) domains. Based on these geometric invariants a preferred coordinate system for the immunoglobulin family is constructed which serves as a basis for structural prediction. The X-ray atom coordinates for all available immunoglobulin structures are transformed to the preferred coordinate system. An affine symmetry between the V(L) and V(H) domains is defined and computed for each of the 53 immunoglobulin structures.     

Penetration and intracellular routing of nucleus-directed peptide-based shuttles (loligomers) in eukaryotic cells

Loligomers are multitasking, peptide-based shuttles that are able to penetrate cells and self-localize into distinct cellular compartments. In particular, loligomer 4 incorporates internalization and nuclear import sequences as well as reporter groups. The intracellular routing of loligomer 4 was analyzed by microscopy and flow cytometry, to define and demonstrate localization events. Electron micrographs of CHO cells exposed to a biotinylated derivative of loligomer 4 as well as confocal images of CHO cells treated with rhodamine-labeled loligomer 4 indicate their presence in the cytosol, endocytic vesicles, and the nucleus of CHO cells. Loligomer 4 accumulates irreversibly inside cells. Uptake of loligomer 4 by six mammalian cell lines (Daudi, EL4, CHO, COS-7, VERO, and HeLa) was proven by flow cytometry, establishing the generality of the principle. Cells presented as monolayers typically were less able to endocytose the construct than cells grown in suspension. Cellular accumulation of loligomer 4 varied between cell lines with COS-7 and VERO cells showing the highest level of uptake. Plasmids harboring reporter genes could be transported efficiently inside CHO cells, suggesting that loligomer 4 either alone or noncovalently associated with large macromolecules can effectively reach the nucleus of cells. In summary, loligomer 4 constructs provide a simple synthetic platform for the design of guided intracellular agents.     

X-Ray diagnosis of pneumothorax in intensive care units (author's transl)

Pneumothorax is the most severe manifestation of pulmonary barotrauma which occurs in mechanical ventilation. Diagnosis of pneumothorax in intensive care radiology is of particular difficulty. Chest radiographs in supine position show a variety of signs which may be helpful but are not conclusive. There are different techniques for verification of ventrally located pneumothorax. 45 degrees tangential radiographs of the hemithorax in question are most conclusive for demonstration of extrapulmonary air located inside the pleural cavity. This 45 degrees technique ist easy to carry out without changing the patients position.     

A study of chromosomes of schistosomiasis patients under oxaminiquine (UK 4271) treatment

Blood samples from 24 patients infested with Schistosoma mansoni were drawn immediately before and 2 days after the administration of a single therapeutic dose of 12-15 mg/kg oral oxaminiquine. Two-day lymphocyte cultures were obtained and about 100 mitoses from each blood sample were analyzed for chromosome aberrations. No significant differences were observed between the "before" and "after" cultures in the frequencies of aberrations resulting from spindle, chromatidic, or chromosome events. It is concluded that there is no reason to fear harmful effects on the chromosomes of patients from treatment with oxaminiquine.     

Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability

Folding stabilities of camelized human antibody VH domains were studied through the determination of their melting points in thermodenaturation experiments. The melting point of a VH domain originating from a synthetic library of human VHs, which had been optimized for the use as small recognition units through the mimicking of camelid antibody heavy chains occurring naturally without light chain, was 56.6 degrees C compared with 71.2 degrees C of the original human VH. Its stability was improved (melting point 61.6 degrees C) through three mutations to mimic camelid VHs even further: Va137 was replaced by phenylalanine and two cysteines were introduced at position 33 and 100b. The resulting VH folded properly and formed a second intradomain disulphide between the extra cysteines. The new mutations were then built constitutively into a phage-display VH library, from which antigen-specific VHs were selected. Two were analysed for stability with melting points of 72.6 and 75.3 degrees C. Thus secondary camelization enabled the isolation of VHs with improved folding stabilities exceeding even that of the original human VH. This indicates an effect on folding stability for some mutations specific in the light chain lacking camelid heavy chains.