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PubMed2025

CCDC137 knockdown suppresses bladder cancer progression by downregulating SCD.

BACKGROUND: The Coiled-coil domain-containing (CCDC) family, due to its unique protein structural domain and broad involvement in diverse biological processes, has emerged as a focus in oncology research. Nevertheless, its clinical significance and function in bladder cancer (BLCA) remain poorly defined. METHODS: Machine learning algorithms were employed to identify pivotal CCDC genes in the cancer genome atlas (TCGA), and a prognostic model was subsequently constructed. Multi-omics data encompassing pan-cancer cohorts, single-cell sequencing, and spatial transcriptomics were integrated to characterize the expression patterns and prognostic significance of Coiled-coil domain-containing 137 (CCDC137), a previously uncharacterized CCDC family member in BLCA. Tissue microarray confirmed CCDC137 abnormal expression in bladder carcinoma specimens. The effect of CCDC137 knockdown on BLCA progression was evaluated through CCK8 assay, clonogenic formation, wound healing, Transwell, and subcutaneous xenograft models. RNA sequencing, quantitative RT-PCR, and western blot were utilized to delineate its regulatory network. RESULTS: A prognostic model incorporating 10 CCDC genes was successfully established in the TCGA-BLCA cohort. Then, we found that CCDC137 exhibited pan-cancer overexpression and usually correlation with poor clinical outcomes. Immunohistochemistry further substantiated its dysregulation in bladder carcinoma. Integrated multi-omics analyses suggested associations between CCDC137 expression and a tumor immunosuppressive microenvironment. CCDC137 knockdown significantly suppressed bladder cancer cell proliferation and migratory capacity in vitro. Correspondingly, subcutaneous xenograft tumor growth was inhibited in vivo. Moreover, decreased expression of stearoyl-CoA desaturase (SCD), a key lipid metabolic enzyme, accompanied CCDC137 depletion. These findings collectively suggest a cancer-promoting role for CCDC137 in bladder carcinoma. CONCLUSIONS: This systematic investigation combining multi-omics bioinformatics analyses and experimental validation demonstrates the role of CCDC137 in bladder carcinoma progression, providing novel mechanistic insights into the pathogenesis of BLCA and offering a theoretical foundation for therapeutic targeting of CCDC137 in urothelial malignancies.

Urinary Bladder Neoplasms↗

PubMed1995

Global regulation of a sigma 54-dependent flagellar gene family in Caulobacter crescentus by the transcriptional activator FlbD.

Biosynthesis of the Caulobacter crescentus polar flagellum requires the expression of a large number of flagellar (fla) genes that are organized in a regulatory hierarchy of four classes (I to IV). The timing of fla gene expression in the cell cycle is determined by specialized forms of RNA polymerase and the appearance and/or activation of regulatory proteins. Here we report an investigation of the role of the C. crescentus transcriptional regulatory protein FlbD in the activation of sigma 54-dependent class III and class IV fla genes of the hierarchy by reconstituting transcription from these promoters in vitro. Our results demonstrate that transcription from promoters of the class III genes flbG, flgF, and flgI and the class IV gene fliK by Escherichia coli E sigma 54 is activated by FlbD or the mutant protein FlbDS140F (where S140F denotes an S-to-F mutation at position 140), which we show here has a higher potential for transcriptional activation. In vitro studies of the flbG promoter have shown previously that transcriptional activation by the FlbD protein requires ftr (ftr for flagellar transcription regulation) sequence elements. We have now identified multiple ftr sequences that are conserved in both sequence and spatial architecture in all known class III and class IV promoters. These newly identified ftr elements are positioned ca. 100 bp from the transcription start sites of each sigma 54-dependent fla gene promoter, and our studies indicate that they play an important role in controlling the levels of transcription from different class III and class IV promoters. We have also used mutational analysis to show that the ftr sequences are required for full activation by the FlbD protein both in vitro and in vivo. Thus, our results suggest that FlbD, which is encoded by the class II flbD gene, is a global regulator that activates the cell cycle-regulated transcription from all identified sigma 54-dependent promoters in the C. crescentus fla gene hierarchy.

Bacterial Proteins↗

PubMed2026

Full-length single-cell spatial transcriptomics reveals spatial and cell-type-specific transcript isoforms in the primate brain.

The primate brain exhibits complex RNA alternative splicing heterogeneity crucial for functional complexity, yet systematic spatial isoform characterization has been lacking. We developed Fullscope-seq, a full-length single-molecule large field-of-view spatial transcriptomics sequencing method at single-cell resolution, based on programmed concatenation cDNA for multiple long-read sequencing platforms. Applying Fullscope-seq to the macaque brain, we uncovered thousands of genes exhibiting differential transcript usage (DTU) across cortical layers, cell types and brain regions. Fullscope-seq resolved hundreds of major isoform switches across distinct brain regions and identified DTUs between superficial and deep cortical layers. Cortical layer-specific DTUs showed cell-composition dependence, whereas regional DTUs were regulated according to both cellular composition and spatial contexts. These isoform variations showed substantial enrichment for neuropsychiatric disorder-associated genes and were conserved across platforms and species. Our study establishes a scalable framework for spatial isoform analysis and provides a resource for understanding transcriptomic diversity in complex tissues.

Animals↗

PubMed1988

Regulation of the penicillinase genes of Bacillus licheniformis: interaction of the pen repressor with its operators.

The synthesis of the inducible enzyme penicillinase of Bacillus licheniformis is negatively controlled by a repressor (D.A. Dubnau and M.R. Pollock, J. Gen. Microbiol. 41:7-21, 1965; D. J. Sherratt and J. F. Collins, J. Gen. Microbiol. 76:217-230,1973). The molecular organization of the genes coding for penicillinase (penP) and its repressor (penI) has recently been determined (T. Himeno, T. Imanaka, and S. Aiba, J. Bacteriol. 168:1128-1132, 1986). These two genes are transcribed divergently from within a 364-nucleotide region separating the coding sequences. We cloned and sequenced the repressor gene (penIc) from strain 749/C that constitutively produces penicillinase. The penIc and penI+ (wild-type) genes were expressed in Escherichia coli. Complementation analysis indicated that the repressor is the only trans-acting protein required to regulate the expression of the penI and penP genes. We purified the wild-type repressor protein, used it in gel retardation and DNase I protection experiments, and identified three operators positioned in the region between the penP and penI coding sequences. The spatial arrangement of the operators and the hierarchy in repressor binding seen in the protection experiments indicate that (i) the penI gene product represses the expression of the penP gene by physically blocking the RNA polymerase-binding site and (ii) the penI gene is autoregulated.

Alleles↗

PubMed2004

Conserved deployment of genes during odontogenesis across osteichthyans.

Odontogenesis has only been closely scrutinized at the molecular level in the mouse, an animal with an extremely restricted dentition of only two types and one set. However, within osteichthyans many species display complex and extensive dentitions, which questions the extent to which information from the mouse is applicable to all osteichthyans. We present novel comparative molecular and morphological data in the rainbow trout (Oncorhynchus mykiss) that show that three genes, essential for murine odontogenesis, follow identical spatial-temporal expression. Thus, at all tooth bud sites, epithelial genes Pitx-2 and Shh initiate the odontogenic cascade, resulting in dental mesenchymal Bmp-4 expression, importantly, including the previously unknown formation of replacement teeth. Significantly, this spatial-temporal sequence is the same for marginal and lingual dentitions, but we find notable differences regarding the deployment of Pitx-2 in the developing pharyngeal dentition. This difference may be highly significant in relation to the theory that dentitions may have evolved from pharyngeal tooth sets in jawless fishes. We have provided the first data on operational genes in tooth development to show that the same signalling genes choreograph this evolutionary stable event in fishes since the osteichthyan divergence 420 Myr ago, with the identical spatial-temporal expression as in mammals.

Animals↗

PubMed1993

CURVATURE: software for the analysis of curved DNA.

Software is presented to plot the sequence-dependent spatial trajectory of the DNA double helix and/or distribution of curvature along the DNA molecule. The nearest-neighbor wedge model is implemented to calculate overall DNA path using local helix parameters: helix twist angle, wedge (deflection) angle and direction (of deflection) angle. The procedures described proved to be very convenient as tools for investigation of a relationship between overall DNA curvature and its gel electrophoretic mobility. All parameters of the model had been estimated from experimental data. Using these wedge parameters the program takes, as input, any DNA sequence and calculates the likely degree of curvature at each point along the molecule. This information is displayed both graphically and in the form of simplified representations of curved double helices. The Software, CURVATURE, can thus be used to investigate possible roles of curvature in modulation of gene expression and for location of curved portions of DNA, which may play an important role in sequence-specific protein--DNA interactions.

Algorithms↗

PubMed1997

Lateral pharyngeal wall motion during swallowing using real time ultrasound.

B-mode ultrasound imaging has been used primarily to detect temporal and spatial movements of the tongue during the oral preparatory and oral stages of swallowing. The purpose of this study was to investigate the application of M-mode (motion mode) ultrasound imaging as a method to quantify the duration and displacement of single regions along the lateral pharyngeal wall during swallows of two bolus volumes and during three swallow maneuvers (supraglottic, super-supraglottic and Mendelsohn maneuver). In 5 normal subjects, simultaneous B/M-mode images were captured at two regions along the lateral pharyngeal wall. Computer-assisted video analysis of each swallow sequence provided spatial coordinates and durational measures. Results indicated no significant differences in displacements of the lateral pharyngeal wall across bolus volumes, swallow maneuvers, or recording sites. Significant differences (p < 0.001) in lateral pharyngeal wall duration occurred as a function of volitional swallow maneuvers. Greater durations (p < 0.05) were found for the Mendelsohn and super-supraglottic swallow maneuvers. The data demonstrate that B/M-mode ultrasound imaging provides a simple, noninvasive method to visually examine movements of the lateral pharyngeal wall and may provide a clinical method for assessing the effects of direct swallowing therapies at the level of the mid-oropharynx.

Adult↗

PubMed1996

Complexity affects regional cerebral blood flow change during sequential finger movements.

Brain regions activated with complex sequential finger movements were localized by measuring regional cerebral blood flow (rCBF) with positron emission tomography. Whereas the total number and frequency of finger movements were kept constant, the complexity of auditory cued sequential finger movements of the right hand varied, with sequence length as the independent variable. In four conditions of differing complexity, the bilateral primary sensorimotor area, left ventral premotor cortex, posterior supplementary motor area, right superior part of the cerebellum, and left putamen were consistently and equally activated. This finding suggests an executive role in running sequences, regardless of their length. The right dorsal premotor cortex (Brodmann area 6) and the right precuneus (Brodmann area 7) showed a linear increase of rCBF as sequence complexity increased. This finding is consistent with the hypothesis that these areas function in the storage of motor sequences in spatial working memory and the production of ongoing sequential movement with reference to that of buffered memory. A similar increase in the cerebellar vermis and the left thalamus likewise suggests a role of these subcortical structures in complexity of sequential finger movements. Conversely, the left inferior parietal lobule showed a decrease of rCBF as complexity increased. Because short-term phonological storage is localized to this area, we suggest that the visuospatial working memory system may suppress other systems not in use. Our findings suggest that complex sequential finger movements recruit a discrete set of brain areas, in addition to areas underlying the execution of simple movement sequences.

Adult↗

PubMed2005

A combinatorial code for splicing silencing: UAGG and GGGG motifs.

Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5'-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes.

Alternative Splicing↗

PubMed2001

Limb apraxia: cortical or subcortical.

Ideomotor apraxia is defined as a disturbance in timing, sequencing, and spatial organization of gestural movements. Left hemisphere motor dominance reflected by ideomotor apraxia mainly refers to spatially and temporally complex movements performed outside the natural context. While clinicoanatomical studies have failed to unveil a specific lesion correlating with apraxia, white matter damage-interrupting corticocortical and corticosubcortical connections-seems crucial for the deficit to be persistent and severe. Patients with basal ganglia lesions and disorders, such as Parkinson's disease and progressive supranuclear palsy, may exhibit ideomotor apraxia. The putative roles of the basal ganglia in object-oriented action, and therefore in praxis, would include among others (a) the selection of the kinematic parameters and the direction of arm movements, (b) working as an integral part of brain systems involved in timing and representation of action sequences, (c) encoding behavioral context, and (d) working as a subcortical component of the parietofrontal circuits devoted to sensorimotor transformation (e.g., reaching). Several studies suggest that basal ganglia pathology per se may not cause overt apraxia. However, when it is combined with dysfunction of the cortical components of the neural systems involved in sequencing, sensorimotor transformation, and response selection, different types of ideomotor praxis deficits would become clinically manifested.

Apraxia, Ideomotor↗

PubMed1997

Tactile pattern perception by two fingers: temporal interference and response competition.

The identification of a spatial pattern (target) presented to one fingerpad may be interfered with by the presentation of a second pattern (nontarget) to either the same fingerpad or a second fingerpad. A portion of the interference appears to be due to masking and a portion to response competition. In the present study, vibrotactile spatial patterns were designed to extend over two fingerpads. Target and nontarget patterns were presented to the same two fingerpads with a temporal separation between the two patterns. The function relating target identification to the temporal separation between the target and nontarget was very similar to the functions obtained with one-finger patterns in temporal masking studies. Subsequent measurements showed that a substantial portion of the interference resulted from response competition. Pattern categorization was better when patterns were presented to two fingers on opposite hands than to two fingers on the same hand; however, there was more interference for patterns presented bilaterally than for patterns presented ipsilaterally. The results supported the conclusion that similar processes are involved in the perception of sequences of spatial patterns whether the patterns are presented to one or to two fingers.

Adult↗

PubMed1998

The Marshall R. Urist Young Investigator Award. Gene expression during autograft lumbar spine fusion and the effect of bone morphogenetic protein 2.

A prospective animal study of posterolateral lumbar spine arthrodesis was performed to determine the temporal and spatial pattern of gene expression and to determine the effect of recombinant human bone morphogenetic protein 2 on the gene expression pattern of a healing spine fusion mass. In Group 1, 20 adult New Zealand rabbits underwent L4-L5 posterolateral intertransverse process arthrodesis using autograft alone. Two rabbits were euthanized at each of the following points: 0, 2, and 4 days, and 1, 2, 3, 4, 5, 6, and 10 weeks after surgery. The same surgical technique was used for 16 rabbits in Group II, except that the autograft first was soaked in a solution of recombinant human bone morphogenetic protein 2 before implantation. Ribonucleic acid was extracted from different regions of the fusion mass at each point and analyzed for expression of bone and cartilage related genes using reverse transcription polymerase chain reaction. A reproducible temporal sequence and spatial pattern of gene expression was found in healing spine fusions. In the central portion of the fusion mass a temporal lag in gene expression was observed that parallels the lag in healing within the central zone previously observed in histologic studies. Treatment of bone graft with recombinant human bone morphogenetic protein 2 resulted in an increase in the early expression of bone morphogenetic protein 6 which was associated with expression of higher levels of Type I collagen, osteocalcin, and other bone related genes. These findings suggest that central nonunion may be associated with delayed expression of osteoblast related genes in the central region of the forming fusion mass. The growth factor, recombinant human bone morphogenetic protein 2, increased the level of bone related gene expression throughout the fusion mass, eliminated the delay in healing within the central zone, and may decrease the likelihood of a nonunion.

Animals↗

PubMed2004

Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity.

RNA silencing regulates gene expression through mRNA degradation, translation repression and chromatin remodelling. The fundamental engines of RNA silencing are RISC and RITS complexes, whose common components are 21-25 nt RNA and an Argonaute protein containing a PIWI domain of unknown function. The crystal structure of an archaeal Piwi protein (AfPiwi) is organised into two domains, one resembling the sugar-binding portion of the lac repressor and another with similarity to RNase H. Invariant residues and a coordinated metal ion lie in a pocket that surrounds the conserved C-terminus of the protein, defining a key functional region in the PIWI domain. Furthermore, two Asp residues, conserved in the majority of Argonaute sequences, align spatially with the catalytic Asp residues of RNase H-like catalytic sites, suggesting that in eukaryotic Argonaute proteins the RNase H-like domain may possess nuclease activity. The conserved region around the C-terminus of the PIWI domain, which is required for small interfering RNA (siRNA) binding to AfPiwi, may function as the receptor site for the obligatory 5' phosphate of siRNAs, thereby specifying the cleavage position of the target mRNA.

Amino Acid Sequence↗

PubMed2000

A method of measuring three-dimensional scapular attitudes using the optotrak probing system.

OBJECTIVE: To develop a method to obtain accurate three-dimensional scapular attitudes and to assess their concurrent validity and reliability. STUDY DESIGN: In this methodological study, the three-dimensional scapular attitudes were calculated in degrees, using a rotation matrix (cyclic Cardanic sequence), from spatial coordinates obtained with the probing of three non colinear landmarks first on an anatomical model and second on a healthy subject. BACKGROUND: Although abnormal movement of the scapula is related to shoulder impingement syndrome, it is not clearly understood whether or not scapular motion impairment is a predisposing factor. Characterization of three-dimensional scapular attitudes in planes and at joint angles for which sub-acromial impingement is more likely to occur is not known. METHODS: The Optotrak probing system was used. An anatomical model of the scapula was built and allowed us to impose scapular attitudes of known direction and magnitude. A local coordinate reference system was defined with three non colinear anatomical landmarks to assess accuracy and concurrent validity of the probing method with fixed markers. Axial rotation angles were calculated from a rotation matrix using a cyclic Cardanic sequence of rotations. The same three non colinear body landmarks were digitized on one healthy subject and the three dimensional scapular attitudes obtained were compared between sessions in order to assess the reliability. RESULTS AND CONCLUSIONS: The measure of three dimensional scapular attitudes calculated from data using the Optotrak probing system was accurate with means of the differences between imposed and calculated rotation angles ranging from 1.5 degrees to 4.2 degrees. Greatest variations were observed around the third axis of the Cardanic sequence associated with posterior-anterior transverse rotations. The mean difference between the Optotrak probing system method and fixed markers was 1.73 degrees showing a good concurrent validity. Differences between the two methods were generally very low for one and two direction displacements and the largest discrepancies were observed for imposed displacements combining movement about the three axes. The between sessions variation of three dimensional scapular attitudes was less than 10% for most of the arm positions adopted by a healthy subject suggesting a good reliability. The Optotrak probing system used with a standardized protocol lead to accurate, valid and reliable measures of scapular attitudes. RELEVANCE: Although abnormal range of motion of the scapula is often related to shoulder pathologies, reliable outcome measures to quantify three-dimensional scapular motion on subjects are not available. It is important to establish a standardized protocol to characterize three-dimensional scapular motion on subjects using a method for which the accuracy and validity are known. The method used in the present study has provided such a protocol and will now allow to verify to what extent, scapular motion impairment is linked to the development of specific shoulder pathologies.

Biomechanical Phenomena↗

PubMed1998

Immunorecognition, ultrastructure and phosphorylation status of astrocytic gap junctions and connexin43 in rat brain after cerebral focal ischaemia.

Gap junctions between astrocytes support a functional syncytium that is thought to play an important role in neural homeostasis. In order to investigate regulation of this syncytium and of connexin43 (Cx43), a principal astrocytic gap junction protein, we determined the sequelae of gap junction and Cx43 disposition in a rat cerebral focal ischaemia model with various ischaemia/reperfusion times using sequence-specific anti-Cx43 antibodies (designated 13-8300, 18A, 16A and 71-0700) that exhibit differential recognition of Cx43, perhaps reflecting functional aspects of gap junctions. Antibody 13-8300 specifically detects only an unphosphorylated form of Cx43 in both Western blots and tissue sections. In hypothalamus after brief (15 min) ischaemic injury, Cx43 at intact gap junctions undergoes dephosphorylation, accompanied by reduced epitope recognition by antibodies 16A and 71-0700. Tissue examined 24 h after reperfusion showed that these effects were reversible. Astrocytic gap junction internalization occurring 1 h after ischaemia was accompanied by decreased immunodetection with 13-8300. At this time, gap junctions were absent in the ischaemic core, coinciding with a loss of Cx43 recognition with 18A and 13-8300, but elevated labelling of internalized Cx43 with 16A and 71-0700. Unphosphorylated Cx43 persisted at intact gap junctions confined to a thin corridor at the ischaemic penumbra which contained presumptive apoptotic cell profiles. Similar results were obtained in ischaemic striatum and cerebral cortex, though with a delayed time course that depended on the severity of the ischaemic insult. These results demonstrate that astrocytic Cx43 epitope masking, dephosphorylation and cellular redistribution occur after ischaemic brain injury, proceed as a temporally and spatially ordered sequence of events and culminate in differential patterns of Cx43 modification and sequestration at the lesion centre and periphery. These observations suggest an attempt by astrocytes in the vicinity of injury to remodel the junctional syncytium according to altered tissue homeostatic requirements.

Animals↗

PubMed1987

Seven structurally different murine monoclonal galactan-specific antibodies show identity in their galactosyl-binding subsite arrangements.

The constants of association of seven monoclonal antibodies--each capable of binding a tetrasaccharide fragment of a linear beta(1,6)-D-galactopyranan--were measured with a series of galactosyl-ligands some of which carried deoxy-fluoro groups at selected locations. In these oligosaccharide ligands, the galactosyl residues bearing a fluorine-instead of a hydroxyl-group, cannot bind to the highest-binding subsite, which requires hydrogen-bonding. This forces a shift in the saccharide contact-residues, and in this way the relative affinities of the antibody subsites for individual galactosyl residues could be evaluated and compared with those of the four subsites investigated earlier. Correlation of sequence data, spatial structure of J 539 and binding behaviour leads to the exclusion of the third complementarity determining region (CDR) of the H-chain as partaking in the binding, and shows that the galactopyranan antigen probably binds along the lower periphery of the H-L interface of the antibodies, and does so in a groove-type fashion. Each of the seven antibodies has four subsites C, A, B and D in going from the H-to the L-chain, and the relative affinity for "their" galactosyl residue decreases in the order A greater than B greater than C greater than D. The single sugar-binding subsite A accounts for ca 50% of the total binding free energy of the maximally binding tetrasaccharide determinant in all cases.

Amino Acid Sequence↗

PubMed2026

Hypoxia Response Is Associated with Reduced HPV Activity and Tumor Microenvironment Remodeling in Cervical Cancer.

Human papillomavirus (HPV) significantly influences cervical cancer progression and treatment, yet its interactions with the tumor microenvironment remain incompletely understood. We performed single-cell and spatial transcriptomic sequencing on cervical cancer samples to explore these interactions. By aligning sequencing reads to a merged HPV16-human reference genome, we characterized HPV16 heterogeneity and its association with host states at the single-cell and single-gene levels. E5 transcriptional activity was negatively associated with the host interferon response, indicating a role in immune evasion. A hypoxic environment was correlated with the downregulation of E5 activity and elevated MHC-I expression, which may contribute to stronger interactions between hypoxic cancer cells and cytotoxic CD8⁺ T cells. Additionally, HPV16 integration in host cells was associated with increased fatty acid metabolism. These findings suggest that combining anti-angiogenic drugs and fatty acid metabolism inhibitors has the potential to improve cervical cancer treatment.

Cervical cancer↗

PubMed1996

Analysis of functional domain organization in DNA topoisomerase II from humans and Saccharomyces cerevisiae.

The functional domain structure of human DNA topoisomerase IIalpha and Saccharomyces cerevisiae DNA topoisomerase II was studied by investigating the abilities of insertion and deletion mutant enzymes to support mitotic growth and catalyze transitions in DNA topology in vitro. Alignment of the human topoisomerase IIalpha and S. cerevisiae topoisomerase II sequences defined 13 conserved regions separated by less conserved or differently spaced sequences. The spatial tolerance of the spacer regions was addressed by insertion of linkers. The importance of the conserved regions was assessed through deletion of individual domains. We found that the exact spacing between most of the conserved domains is noncritical, as insertions in the spacer regions were tolerated with no influence on complementation ability. All conserved domains, however, are essential for sustained mitotic growth of S. cerevisiae and for enzymatic activity in vitro. A series of topoisomerase II carboxy-terminal truncations were investigated with respect to the ability to support viability, cellular localization, and enzymatic properties. The analysis showed that the divergent carboxy-terminal region of human topoisomerase IIalpha is dispensable for catalytic activity but contains elements that specifically locate the protein to the nucleus.

Amino Acid Sequence↗