Final Step In Formation Research Articles

Binding of the Treslin-MTBP Complex to Specific Regions of the Human Genome Promotes the Initiation of DNA Replication

SUMMARYThe processes that control where higher eukaryotic cells initiate DNA replication throughout the genome are not understood clearly. In metazoans, the Treslin-MTBP complex mediates critical final steps in formation of the activated replicative helicase prior to initiation of replication. Here, we map the genome-wide distribution of the MTBP subunit of this complex in human cells. Our results indicate that MTBP binds to at least 30,000 sites in the genome. A majority of these sites reside in regions of open chromatin that contain transcriptional-regulatory elements (e.g., promoters, enhancers, and super-enhancers), which are known to be preferred areas for initiation of replication. Furthermore, many binding sites encompass two genomic features: a nucleosome-free DNA sequence (e.g., G-quadruplex DNA or AP-1 motif) and a nucleosome bearing histone marks characteristic of open chromatin, such as H3K4me2. Taken together, these findings indicate that Treslin-MTBP associates coordinately with multiple genomic signals to promote initiation of replication.

Unexpected Accumulation of ncm5U and ncm5s2U in a trm9 Mutant Suggests an Additional Step in the Synthesis of mcm5U and mcm5s2U

BackgroundTransfer RNAs are synthesized as a primary transcript that is processed to produce a mature tRNA. As part of the maturation process, a subset of the nucleosides are modified. Modifications in the anticodon region often modulate the decoding ability of the tRNA. At position 34, the majority of yeast cytosolic tRNA species that have a uridine are modified to 5-carbamoylmethyluridine (ncm5U), 5-carbamoylmethyl-2′-O-methyluridine (ncm5Um), 5-methoxycarbonylmethyl-uridine (mcm5U) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The formation of mcm5 and ncm5 side chains involves a complex pathway, where the last step in formation of mcm5 is a methyl esterification of cm5 dependent on the Trm9 and Trm112 proteins.Methodology and Principal FindingsBoth Trm9 and Trm112 are required for the last step in formation of mcm5 side chains at wobble uridines. By co-expressing a histidine-tagged Trm9p together with a native Trm112p in E. coli, these two proteins purified as a complex. The presence of Trm112p dramatically improves the methyltransferase activity of Trm9p in vitro. Single tRNA species that normally contain mcm5U or mcm5s2U nucleosides were isolated from trm9Δ or trm112Δ mutants and the presence of modified nucleosides was analyzed by HPLC. In both mutants, mcm5U and mcm5s2U nucleosides are absent in tRNAs and the major intermediates accumulating were ncm5U and ncm5s2U, not the expected cm5U and cm5s2U.ConclusionsTrm9p and Trm112p function together at the final step in formation of mcm5U in tRNA by using the intermediate cm5U as a substrate. In tRNA isolated from trm9Δ and trm112Δ strains, ncm5U and ncm5s2U nucleosides accumulate, questioning the order of nucleoside intermediate formation of the mcm5 side chain. We propose two alternative explanations for this observation. One is that the intermediate cm5U is generated from ncm5U by a yet unknown mechanism and the other is that cm5U is formed before ncm5U and mcm5U.

0340: 3D Ultrasound of the Upper Abdomen

The digital revolution has made three-dimensional (3D) ultrasonography a natural extension of 2D ultrasound scanning. This lecture reviews some of the principles and methods in 3D ultrasonography of the upper abdomen. At present, the process of making 3D images based on ultrasonography is commonly divided into 5 steps: Data acquisition, data digitization, data storage, data processing, and data display. Principally, data acquisition by 3D ultrasonography can be performed in 3 different ways; either by using a 2D probe attached to a motor which moves the probe in a computer-defined way or by a spatial localizing system connected to a 2D probe or by genuine, electronic 3D probes with the possibility of direct volume acquisition in real time. True volumetric 2D array transducers instantly generate a volume of ultrasound data, enabling dynamic 3D ultrasonography. A critical step in processing of 3D data is segmentation, which is the procedure where the object of interest is separated from the surrounding structures. Three fundamental approaches to segmentation have been utilised in 3D ultrasonography: Extraction by visual inspection and manual outlining of contours; semi-automatic separation using visualisation algorithms aided by operator interaction, and fully automatic computer segmentation. The final step in formation of 3D ultrasound images is to display the data so that the inherent voxel information is communicated accurately. Commonly, simple rotation of the object on the computer monitor provides some 3D effects. To further enhance the 3D outcome of the images, stereoglasses can be applied. Data processing and display requires specialized computer software to handle the ultrasound images. Ultrasound data contains a significant amount of noise and speckle and may exhibit boundary regions several pixels wide. It is important to keep in mind that the quality of the final 3D data display strongly depends on the resolution of the raw data. Transducer frequency and lateral resolution, frame rate of the scanner, accuracy of 3D probe, speed of scanning, methods of filtering and segmentation, are all factors that influences the final 3D image and subsequently accuracy in volume measurements.

Changes in alpha (1-2)-fucosyltransferase activity in the murine endometrial epithelium during the estrous cycle, early pregnancy, and after ovariectomy and hormone replacement.

alpha (1-2)-Fucosyltransferase (alpha (1-2)-FT) activity in the uterine luminal epithelium was investigated during the estrous cycle, during early pregnancy, and in ovariectomized mice treated with ovarian steroids. Phenyl-beta-D-galactoside was used as a specific acceptor for alpha (1-2)-FT activity. Enzyme activity fell dramatically throughout the pre- and peri-implantation period (Days 1-6 of pregnancy). Activity on Day 4 of pregnancy represented less than a tenth of the Day 1 level and by Day 6 became undetectable. During the estrous cycle alpha (1-2)-FT showed a peak of activity at estrus, the level being similar to that observed on Day 1 of pregnancy. In proestrus, postestrus, and diestrus, activity was similar and was approximately one fifth of that during estrus. Ovariectomized mice were primed with estrogen and treated with progesterone only, estradiol benzoate only, or progesterone followed by a single injection of progesterone and estradiol. Estrogen alone stimulated alpha (1-2)-FT activity, while progesterone treatment was strongly inhibitory. These results correlate well with the pattern of expression of the fucosylated H-type-1 carbohydrate epitope found on the endometrial epithelium and believed to have a role in implantation. alpha (1-2)-FT is capable of catalyzing the final step in formation of the H-type-1 oligosaccharide, and on the basis of our results it is likely that control of this enzyme is the major factor responsible for modulating H-type-1 expression.

Structure-activity relationship of polyamine derivatives of 1,3-dichloroacetone-thiosemicarbazone: Induction of metastases and increase in sialylation of murine lymphoma L5178Y-R cells

A number of polyamine (PA) derivatives of thiosemicarbazone of 1,3-dichloroacetone (TDA) have been prepared and their effect on growth in vivo of tumorigenic but not metastatic cell strain (LY-R) of mouse lymphoma L5178Y has been investigated. Polyamine derivatives of TDA (PDT) were injected i.p. every third day (4 times, 10 or 25 mg/kg per injection) into DBA/2 mice inoculated i.p. or s.c. with LY-R cells. It has been found that disubstituted putrescine (Put), spermidine (Spd) and spermine (Spm) derivatives TDA exhibit a prometastatic activity as indicated by the appearance of solid tumor foci in subcutaneous tissues, liver and spleen. This activity depends mainly on the structure of the PA fragment and the presence of TDA. An increase in lipid bound sialic acid content after treating LY-R cells in vitro and in vivo with a Spm derivative has been found. These findings suggest that disubstituted PA derivatives of TDA and LY-R cells may be a useful model for investigation of the final steps in formation of metastases by lymphoma cells.

THE ACTIVATION OF CHYMOTRYPSINOGEN: ISOLATION AND IDENTIFICATION OF A PEPTIDE LIBERATED DURING ACTIVATION

Reactions attending the conversion of precursors of proteolytic enzymes to the active form are distinguished by several features of unusual interest: (a) they are enzyme-catalyzed; (b) they are irreversible; (c) they involve the opening of a limited number of peptide bonds; (d) they provide an experimental system for the study of the final steps in formation of a biologically specific protein and of the relation of chemical structure to biological activity of proteins. In recent studies in this laboratory, including the present work, several intermediates formed during the activation of chymotrypsinogen have been recognized by a combination of methods. These have included electrophoresis, end-group analysis, enzymatic activity, peptide analysis, and sedimentation. As a result of the present experimental study, which dealt in particular with the nature of the peptide released during the rapid activation of chymotrypsinogen, it has been possible to identify the nature of the limited number of peptide bonds hydrolyzed during the activation process.