A polymorphism in the NPPA gene associates with asthma.

BACKGROUND: Atrial natriuretic peptide (ANP) plays an important role in the lung and in augmenting allergic inflammation in asthma. The gene encoding ANP, NPPA, is located on chromosome 1p36, a region that has been linked to asthma. OBJECTIVES: Determine associations between asthma and four common SNPs on the NPPA gene: C/G (rs13305986) in the promoter; G/A (rs5063) in Exon 1 resulting in NPPAMet32-->Val substitution; T/C (rs5065) in Exon 3 resulting in an Arg152-->Ter substitution; and T/C in the 3'UT region (rs5067). Methods A case-control design was used in White participants. The screening cohort consisted of 336 asthmatic cases who participated in a large clinical trial and 154, non-asthmatic controls. The replicate cohort consisted of 172 asthmatic cases from a second clinical trial and 115 healthy controls. Demographic characteristics were well matched for cases and controls in the screening cohort. Adjusted (age, gender, body mass index) odds ratio (OR) were calculated by chi(2) and logistic regression; a P-value of 0.0167 defined the threshold of significance. RESULTS: The C allele of rs5067 was associated with asthma in the screening and replicate cohorts: adjusted ORs (95% confidence intervals) 0.5 (0.29-0.84; P=0.009) and 0.24 (0.11-0.53; P<0.0001), respectively. The C allele of rs5065 was associated with asthma in the screening cohort but not in the replicate. The population-attributable risk for asthma in carriers of the C allele for rs5067 was 23.3%. CONCLUSIONS: For rs5067, the risks of asthma in carriers of the C allele in the screening and replicate cohorts were reduced by 50% and 76%, respectively. NPPA may be an important susceptibility gene for asthma.

Low exhaled nitric oxide and a polymorphism in the NOS I gene is associated with acute chest syndrome.

Abnormalities of nitric oxide metabolism have been implicated in the pathogenesis of acute chest syndrome in subjects with sickle cell anemia. It is not known whether exhaled nitric oxide levels (FE(NO)) are abnormal in children with a history of the acute chest syndrome (ACS). We compared FE(NO), plasma nitric oxide metabolites (NO(x)), serum arginine and citrulline levels, and the number of AAT repeats in intron 20 of NOS I in subjects with sickle cell disease (SCD) and a history of at least one episode of ACS (ACS(+), n = 13), subjects with SCD and no prior history of ACS (ACS(-), n = 7), and healthy children (HC, n = 6). Mean +/- SD FE(NO) (ppb) was lower in ACS(+) than in ACS(-) and HC: (10.4 +/- 4.3 versus 23.4 +/- 6.1 p = 0.002] and 30.4 +/- 15.8 [p = 0.0001], respectively). Plasma NO(x) (microM) were similar in all three groups (37.3 +/- 19.4, 33.0 +/- 13.2, 44.7 +/- 7.8, respectively). Arginine and citrulline levels (microM) did not differ between ACS(+) and ACS(-) groups. Spirometric data revealed a mildly diminished FEV(1) and FVC in ACS(+) that was statistically different from HC but not ACS(-): (FEV(1) as % of predicted for ACS(+), ACS(-), and HC; 83 +/- 17 versus 87 +/- 16 versus 102 +/- 16, respectively, p < 0.05 between ACS(+) and HC). The level of FE(NO) was significantly associated with the sum of AAT repeats in intron 20 of NOS I gene alleles. The correlation coefficient (r) was 0.62 (p < 0.005). We conclude that FE(NO) levels are significantly reduced in subjects who have a history of ACS and that the FE(NO) levels are significantly correlated with the number of NOS I AAT repeats. FE(NO) is a sensitive marker and may be a predictor of ACS prone children.

10-year biochemical (prostate-specific antigen) control of prostate cancer with (125)I brachytherapy.

PURPOSE: To report 10-year biochemical (prostate-specific antigen [PSA]) outcomes for patients treated with 125I brachytherapy as monotherapy for early-stage prostate cancer. METHODS AND MATERIALS: One hundred and twenty-five consecutively treated patients, with clinical Stage T1-T2b prostate cancer were treated with 125I brachytherapy as monotherapy, and followed with PSA determinations. Kaplan-Meier estimates of PSA progression-free survival (PFS), on the basis of a two consecutive elevations of PSA, were calculated. Aggregate PSA response by time interval was assessed. Comparisons were made to an earlier-treated cohort. RESULTS: The overall PSA PFS rate achieved at 10 years was 87% for low-risk patients (PSA < 10, Gleason Sum 2-6, T1-T2b). Of 59 patients (47%) followed beyond 7 years, 51 (86%) had serum PSAs less than 0.5 ng/mL; 48 (81%) had serum PSAs less than 0.2 ng/mL. Failures were local, 3.0%; distant, 3.0%. No patients have died of prostate carcinoma. The proportion of patients with a PSA < or =0.2 ng/mL continued to increase until at least 7-8 years posttherapy. A plot of PSA PFS against the proportion of patients achieving serum PSA of less than 0.2 ng/mL suggests a convergence of these two endpoints at 10 years. Patients treated in the era of this study (1988-1990) experienced a statistically improved PFS compared with an earlier era (1986-1987). This difference appears independent of patient selection, suggesting that the maturation of the technique resulted in improved biochemical control. CONCLUSION: With modern technique, monotherapy with 125I achieves a high rate (87%) of biochemical and clinical control in patients with low-risk disease at 10 years. The decline of PSA following brachytherapy with low-dose-rate isotopes can be protracted. Absolute PSA and PFS curves merge, and are comparable at 10 years.

Human rDNA: evolutionary patterns within the genes and tandem arrays derived from multiple chromosomes.

Human rDNA forms arrays on five chromosome pairs and is homogenized by concerted evolution through recombination and gene conversion (loci RNR1, RNR2, RNR3, RNR4, RNR5, OMIM: 180450). Homogenization is not perfect, however, so that it becomes possible to study its efficiency within genes, within arrays, and between arrays by measuring and comparing DNA sequence variation. Previous studies with randomly cloned genomic DNA fragments showed that different parts of the gene evolve at different rates but did not allow comparison of rDNA sequences derived from specific chromosomes. We have now cloned and sequenced rDNA fragments from specific acrocentric chromosomes to (1) study homogenization along the rDNA and (2) compare homogenization within chromosomes and between homologous and nonhomologous chromosomes. Our results show high homogeneity among regulatory and coding regions of rDNA on all chromosomes, a surprising homogeneity among adjacent distal non-rDNA sequences, and the existence of one to three very divergent intergenic spacer classes within each array.

Arrangement of ribosomal genes in nucleolar domains revealed by detection of "Christmas tree" components.

We investigated how the transcribing ribosomal genes ("Christmas trees") of HeLa cells are arranged in the nucleolus. Hypotonic conditions let the granular component disperse, while fibrillar centres and parts of the dense fibrillar component were resistant to low ionic strength conditions. Both remained within the former nucleolar territory. We used immunocytochemistry and in situ hybridisation at the light microscopic and ultrastructural level for the analysis of the internal nucleolar structures. The 5' ends of ribosomal RNA and ribosomal DNA sequences were found associated with the periphery of fibrillar centres. The hypotony-resistant parts of the dense fibrillar component did not contain the 5' end of the transcript or the gene. The downstream ribosomal DNA sequences were found in the nucleolar territory but not associated with any hypotony-resistant structures. The downstream ribosomal RNA revealed a similar distribution. We show that transcription initiation and transcript elongation occur in different molecular and structural environments. Transcription initiation is located at the periphery of fibrillar centres. Evidently the dense fibrillar component is non-homogeneous in molecular composition. Transcript elongation is continued in a part of the dense fibrillar component which is dissolved under intermediate hypotonic conditions. A structural model of nucleolar transcription is suggested.

Determination of human beta(2)-adrenoceptor haplotypes by denaturation selective amplification and subtractive genotyping.

OBJECTIVE: beta(2)-Adrenoceptor haplotype may be better associated with asthma severity and drug response than a polymorphic variant at any single site. Because present methods of haplotype determination are time consuming and impractical for large population studies, we sought to develop a simple and efficient method of determining haplotype of 3 common polymorphisms at codons -19 (Arg/Cys), 16 (Arg/Gly) and 27 (Glu/Gln). DESIGN: Preliminary studies showed that the C/G base pair of the Arg(-19) allele increases the local melting temperature over the T/A base pair of the Cys(-19) allele by 3.6 degrees C and establishes a new local maximum denaturation temperature. By choosing a suitable denaturation temperature and appropriate primers and coupling them with restriction fragment length polymorphism (RFLP) analysis, we hypothesized that the genotype of one separately amplified allele followed by subtraction from the combined genotype of two alleles would yield the beta(2) haplotype in > 99% of the population. RESULTS: Haplotype determined by our method was in complete agreement with haplotype determined by cloning and sequencing in 29 samples. The frequencies of haplotype pairs in 78 healthy adults, according to our method, were in agreement with published values that were inferred, and were: RGE/CRQ, 26.9%; CRQ/CRQ, 25.6%; RGE/CGQ, 16.7%; CRQ/CGQ, 10.3%; RGE/RGE, 11.5%; CGQ/CGQ, 7.7%. The haplotype pair in one individual was RRE/CRQ (1.3%). CONCLUSION: Our method of determining beta(2)-adrenoceptor haplotype is simple, accurate and cost effective for haplotyping large populations.

Karyotypic dissection of Hodgkin's disease cell lines reveals ectopic subtelomeres and ribosomal DNA at sites of multiple jumping translocations and genomic amplification.

Although the neoplastic significance of the chromosome changes widespread in Hodgkin's disease (HD) remains obscure, a distinct cytogenetic picture has emerged combining aneuploidy with structural rearrangements clustered at certain breakpoints. Notably absent are the recurrent chromosome translocations which distinguish other hematopoietic neoplasms and serve as clues to underlying oncogene alterations. The paucity of neoplastic cells in HD biopsies hinders detailed chromosome analysis. As an alternative, we investigated a panel of well characterized cell lines by classical and molecular cytogenetics, using single-gene and subtelomeric probes, including three autologous HD examples (HDLM-1/2/3) analyzed by 'spectral karyotyping' - the first complete HD karyotype to be documented. Although complex, most rearrangements in HDLM cells arose in vivo and included few rare but many typical HD breakpoints, notably at the r(ibosomal)DNA regions. Two types of genomic rearrangement involving DNA repeats were conspicuous: insertion and genomic amplification/coamplification of rDNA-the first genomic rDNA rearrangements to be reported in a tumor cell, and the first example of multiple 'jumping translocations' (JT). Of four subtelomeric microsatellite repeats tested in HDLM cells, three exhibited interstitial sites at JT, of which two (at 5qter and 9pter) were respectively associated with deletion of the 5q31-32 myeloid region, and coamplification of a recently described HD-recurrent amplicon at 9p2 together with transcriptionally silent rDNA. Altogether, three out of four HD cell lines carried interstitial 9p subtelomeres and rDNA rearrangements. Taken together, these data suggest tumorigenic rearrangements may be facilitated by 'hitchhiking' along with mobile DNA repeat sequences which may target gene rearrangement at 9p in HD. Southern analysis of parallel rearrangements within rDNA intergenic spacers in HDLM cells highlighted several at, or near, retroposons. As well as validating HD cell lines as cytogenetic models, and resources for identifying genes rearranged in HD, our findings warrant further investigation of the roles of DNA repeat sequences, notably subtelomeric microsatellites, rDNA spacer sequences and retroposons as facilitators and markers of tumor-gene rearrangement.

Transient elevation of serum prostate-specific antigen following (125)I/(103)Pd brachytherapy for localized prostate cancer.

Based on suggestions by anecdotal evidence to date, an attempt is made to estimate the occurrence of non-disease-related prostate-specific antigen (PSA) spiking in the serum PSA profiles of a series of men treated by (125)I/(103)Pd brachytherapy with or without external beam irradiation. Five hundred ninety-one patients treated between January 1988 and December 1993 were eligible for study. Patients whose clinical status was described as equivocal (declining PSA > 1.0 ng/mL or rising PSA without documented disease [9.6% of the cohort]) were not considered. Evidence of PSA increases that were followed by decline were identified. Treatment and disease-specific parameters were examined for influence of the occurrence of spiking. In patients judged biochemical successes at last follow-up (serum PSA < or = 1.0 ng/mL), 35.8% exhibited a temporary increase of 0.2 ng/mL or more. Seventy-five percent of these patients exhibited a temporary increase between 0.3 and 3.4 ng/mL. The average time of the temporary increases was 24.8 months after implant. Spiking was not associated with a higher risk of clinical failure in this data set. Conventional risk factors for recurrent disease were not associated with benign PSA spiking. Low-magnitude serum PSA spiking may occur in up to one third of patients following permanent, low-dose rate brachytherapy of the prostate. Most of these observations occur up to 3 years after implant and do not appear to be related to disease recurrence. Caution should be taken before initiating further therapy pursuant to the observation of PSA spiking of less than 2 to 3 ng/mL shortly following brachytherapy. Frequent serum PSA sampling following prostate brachytherapy with early follow-up may overestimate biochemical failure rates.

Mammalian mitochondrial ribosomal proteins (4). Amino acid sequencing, characterization, and identification of corresponding gene sequences.

Mitochondrial ribosomal proteins (MRPs) are required for the translation of all 13 mitochondrial encoded genes in humans. It has been speculated that mutations and polymorphisms in the human MRPs may be a primary cause of some oxidative phosphorylation disorders or modulate the severity and tissue specificity of pathogenic mitochondrial DNA mutations. Although the sequences of most of the yeast MRPs are known, only very few mammalian and nearly no human MRPs have been completely characterized. MRPs differ greatly in sequence, and sometimes biochemical properties, between different species, not allowing easy recognition by sequence homology. Therefore, the Mammalian Mitochondrial Ribosomal Consortium is using a direct approach of purifying individual mammalian (bovine) MRPs, determining their N-terminal and/or internal peptide sequences using different protein sequencing techniques, and using the resulting sequence information for screening expressed sequence tags and genomic data bases to determine human, mouse, and rat homologues of the bovine proteins. Two proteins of the large and three proteins of the small ribosomal subunit have been analyzed in this manner. Three of them represent "new," i.e. formerly unknown mammalian mitochondrial ribosomal protein classes. Only one of these three different MRPs shows significant sequence similarities to known ribosomal proteins. In one case, the corresponding human genomic DNA sequences were found in the data bases, and the exon/intron structure was determined.

Palladium-103 brachytherapy for prostate carcinoma.

PURPOSE: A report of biochemical outcomes for patients treated with palladium-103 (Pd-103) brachytherapy over a fixed time interval. METHODS AND MATERIALS: Two hundred thirty patients with clinical stage T1-T2 prostate cancer were treated with Pd-103 brachytherapy and followed with prostate-specific antigen (PSA) determinations. Kaplan-Meier estimates of biochemical failure on the basis of two consecutive elevations of PSA were utilized. Multivariate risk groups were constructed. Aggregate PSA response by time interval was assessed. RESULTS: The overall biochemical control rate achieved at 9 years was 83.5%. Failures were local 3.0%; distant 6.1%; PSA progression only 4.3%. Significant risk factors contributing to failure were serum PSA greater than 10 ng/ml and Gleason sum of 7 or greater. Five-year biochemical control for those exhibiting neither risk factor was 94%; one risk factor, 82%; both risk factors, 65%. When all 1354 PSA determinations obtained for this cohort were considered, the patients with a proportion of PSAs < or = 0.5 ng/ml continued to increase until at least 48 months post-therapy. These data conformed to a median PSA half-life of 96.2 days. CONCLUSIONS: Prostate brachytherapy with Pd-103 achieves a high rate of biochemical and clinical control in patients with clinically organ-confined disease. PSA response following brachytherapy with low-dose-rate isotopes is protracted.

Heart-specific splice-variant of a human mitochondrial ribosomal protein (mRNA processing; tissue specific splicing).

It has been proposed that splice-variants of proteins involved in mitochondrial RNA processing and translation may be involved in the tissue specificity of mitochondrial DNA disease mutations (Fischel-Ghodsian, 1998. Mol. Genet. Metab. 65, 97-104). To identify and characterize the structural components of mitochondrial RNA processing and translation, the Mammalian Mitochondrial Ribosomal Consortium has been formed. The 338 amino acid (aa) residues long MRP-L5 was identified (O'Brien et al., 1999. J. Biol. Chem. 274, 36043-36051), and its transcript was screened for tissue specific splice-variants. Screening of the EST databases revealed a single putative splice-variant, due to the insertion of an exon consisting of 89 nucleotides prior to the last exon. Screening of multiple cDNA libraries revealed this inserted exon to be present only in heart tissue, in addition to the predominant MRP-L5 transcript. Sequencing of this region confirmed the EST sequence, and showed in the splice-variant a termination triplet at the beginning of the last exon. Thus the inserted exon replaces the coding sequence of the regular last exon, and creates a new 353 aa long protein (MRP-L5V1). Sequence analysis and 3D modeling reveal similarity between MRP-L5 and threonyl-t-RNA synthetases, and a likely RNA binding site within MRP-L5, with the C-terminus in proximity to the RNA binding site. Sequence analysis of MRP-L5V1 also suggests a likely transmembrane domain at the C-terminus. Thus it is possible that the MRP-L5V1 C-terminus could interfere with RNA binding and may have gained a transmembrane domain. Further studies will be required to elucidate the functional significance of MRP-L5V1.

Mammalian mitochondrial ribosomal proteins (2). Amino acid sequencing, characterization, and identification of corresponding gene sequences.

Four different classes of mammalian mitochondrial ribosomal proteins were identified and characterized. Mature proteins were purified from bovine liver and subjected to N-terminal or matrix-assisted laser-desorption mass spectroscopic amino acid sequencing after tryptic in-gel digestion and high pressure liquid chromatography separation of the resulting peptides. Peptide sequences obtained were used to virtually screen expressed sequence tag data bases from human, mouse, and rat. Consensus cDNAs were assembled in silico from various expressed sequence tag sequences identified. Deduced mammalian protein sequences were characterized and compared with ribosomal protein sequences of Escherichia coli and yeast mitochondria. Significant sequence similarities to ribosomal proteins of other sources were detected for three out of four different mammalian protein classes determined. However, the sequence conservation between mitochondrial ribosomal proteins of mammalian and yeast origin is much less than the sequence conservation between cytoplasmic ribosomal proteins of the same species. In particular, this is shown for the mammalian counterparts of the E. coli EcoL2 ribosomal protein (MRP-L14), that do not conserve the specific and functional highly important His(229) residue of E. coli and the corresponding yeast mitochondrial Rml2p.

Interstitial iodine-125 radiation without adjuvant therapy in the treatment of clinically localized prostate carcinoma.

BACKGROUND: This study was designed to evaluate the efficacy of iodine-125 interstitial radiation in the treatment of prostate carcinoma classified as T1 or T2. METHODS: One hundred twenty-six consecutive patients with adenocarcinoma of the prostate (T1, 23%; T2, 77%) were treated with iodine-125 radionuclides between January 1, 1988, and December 31, 1990. Four patients died of intercurrent illness within 1 year postimplant, leaving 122 men in the study. The prescribed minimum radiation dose was 160 gray. Median follow-up was 69.3 months. Prebiopsy prostate specific antigen (PSA) values (median, 5.0 ng/mL) were available for all patients. Posttherapy evaluation included clinical, biochemical (PSA), and pathologic (repeat needle biopsy) studies. No patient was surgically staged, and none received androgen deprivation therapy. Morbidity was graded according to the Radiation Therapy Oncology Group grading scale. Statistical appraisal was performed by the Kaplan-Meier method. PSA failure was defined in two ways: (1) PSA progression, i.e., 2 consecutive increases from a nadir value; and (2) failure to attain an arbitrary serum PSA value of 1.0 or 0.5 ng/mL at last follow-up. RESULTS: The overall 7-year survival was 77%; there were no deaths from prostate carcinoma in this cohort. The 7-year actuarial PSA progression free outcome was 89%, and the PSA < or = 1.0 ng/mL outcome was 87%. When PSA < or = 0.5 ng/mL was selected as an outcome end point, and PSA values in this series of radiation-treated patients were compared with PSA values proposed to indicate disease free survival after radical prostatectomy (PSA < or = 0.3-< or = 0.6 ng/mL), the 7-year actuarial disease free survival was 79%. Morbidity was minimal except in patients who had preimplant or postimplant transurethral prostate resection. CONCLUSIONS: Outpatient-based iodine-125 prostate brachytherapy for prostate carcinoma classified as T1 or T2 resulted in biochemical outcomes comparable to end points resulting from radical prostatectomy and external beam radiation.

Beyond ribosomal DNA: on towards the telomere.

We have sequenced and analyzed 8.3 kb of sequence adjacent and distal to the human ribosomal DNA (rDNA); this distal sequence connects to the rDNA cluster just 4 kb upstream of the first promoter and is shared among the acrocentric chromosomes and, at least in part, it is also present in other primates. The sequence differs in character from that of the rDNA intergenic spacer (IGS) in that it does not contain long stretches of either polypyrimidine or polypurine. However, just like the IGS, it contains numerous repetitive elements, including retroposed fragments of 28S rRNA and large pieces of the IGS. In addition, we show that the rDNA clusters are not interrupted by other sequences and do not recombine with this distal segment.

Incognito rRNA and rDNA in databases and libraries.

Both ribosomal DNA (rDNA) and ribosomal RNA (rRNA) are over-represented in the starting material for genomic and cDNA libraries; thus, their sequences have the potential of repeatedly entering the various databases. When DNA (both transcribed and intergenic spacer regions) is used as query sequence, a great number of matches are found in the databases, particularly in the EST database, and to a lesser extent among genomic sequences and STSs, which are not identified as rDNA. We discuss the following explanations for the widespread occurrence of rDNA in cDNA and genomic DNA libraries: pseudogenes of rRNA in other genomic locations, mRNA-derived pseudogenes that reside in rDNA, cDNAs derived from rRNA [either by self-priming or by internal oligo(dT) priming], cDNAs derived from actual transcripts of the rDNA intergenic spacer, and genomic DNA contamination of RNA preparations. Because so many database entries contain unidentified rDNA, we recommend that all sequence submissions be checked (by the submitters) for the presence of structural RNAs in addition to repetitive sequences.

Human ribosomal RNA variants from a single individual and their expression in different tissues.

We have investigated the extent of sequence variation in human ribosomal RNA (rRNA) genes and the expression of specific rRNA gene variants in different tissues of an individual. Focusing on the fifth variable region (V5; nt 2065-2244) of the 28S rRNA gene, we find that sequence differences between rRNA genes of a single individual are characterized by differences in number of repeats of simple sequences at four specific sites. These data support and extend previous findings which show similar V5 sequence variation in rRNA genes from a group of individuals. We performed experiments to determine if there is differential gene expression within the rRNA multigene family. From the analysis of data of six variant V5 probes protected from RNase digestion by rRNAs isolated from different tissues of the individual, we conclude that each variant rRNA is present in a similar proportion in these tissues, whereas the actual contributions of variants differ, their relative proportion is maintained from tissue to tissue in an individual. We favor the explanation of a gene dosage effect over that of a regulated gene effect to account for this pattern of rRNA gene expression. In addition, computer generated secondary structure models of each V5 clone structure predict the same three helix structure with the regions of sequence variation contained in one stem-loop structure.

Should brachytherapy be considered a therapeutic option in localized prostate cancer?

Contemporary prostate brachytherapy incorporates advances in computer analysis, imaging technology, and delivery apparatus, allowing exacting and reproducible results compared with historical approaches. The advances permit brachytherapy to be performed on a cost-effective, outpatient basis with low morbidity in the appropriately selected patient. Although unsettled questions remain regarding dosimetric issues, long-term outcomes, and morbidity, the weight of evidence to date appears to support the use of brachytherapy in selected patients. Brachytherapy may be considered a therapeutic option: as monotherapy for early-stage disease and also a boost following moderate doses of external beam irradiation for locally advanced disease.

The myosin filament XV assembly: contributions of 195 residue segments of the myosin rod and the eight C-terminal residues.

A mixture of two peptides of approximately M(r) 13000 has been isolated from a papain digest of LC2 deficient myosin. The peptides assemble into highly ordered aggregates which in one view are made up of strands of pairs of dots with an average side to side spacing of 13.0 nm and an average axial repeat of 9.0 nm. In another view there are strands of single dots with a side-to-side spacing of 7.8 nm and an axial repeat of 9.1 nm. From N-terminal peptide sequencing, the two peptides have been shown to come from regions of the myosin rod displaced by 195 residues. We have shown that either peptide alone can assemble to form the same aggregates. The 195 residue displacement of the M(r) 13000 peptides corresponds closely to the 196 residue repeat of charges along the myosin rod. This finding permits us to designate 195 residue segments of the myosin rod and to relate assembly characteristics directly to the similar 195 residue segments and 196 residue charge repeat. The most C-terminal 195 residue segment carries information for assembly into helical strands. The contiguous 195 residue segment, in major part, carries information for the unipolar assembly, characteristic of the assembly in each half of the myosin filament. The next contiguous 195 residue segment, in major part, carries information for bipolar assembly which is characteristic of the bare zone region of the filament; and for the transition from the bipolar bare zone to unipolar assembly. The effect of the eight C-terminal residues of the myosin rod on the assembly of the contiguous 195 residues has also been studied. The entire fragment of 195 + eight C-terminal residues assembled to form helical strands with an axial repeat of 30 nm. Successive deletion of charged residues changed the axial repeat of the helical strands suggesting that the charged residues at the C-terminus are involved in determining the pitch in the helical assembly of the contiguous 195 residues.

Topoisomerase-II-mediated DNA cleavage within the human ribosomal genes.

The interaction of topoisomerase II (topo II) with human ribosomal DNA (rDNA) was investigated in vivo using the antitumoral drug VM26, a specific inhibitor of topo II, that stabilizes the transient cleavable complex. rDNA-protein complexes isolated from nucleoli of TG cells were analyzed for double strand breaks with probes that covered almost all intergenic transcribed spacer (IGS) and all transcribed sequences of tandem repeat ribosomal DNA genes. Preferential cleavage sites were present in only a part of nucleolar rDNA, i.e., the transcribed region. Proteins, purified from the same complexes, were analyzed by Western-blot and stained by an antiserum against both topo II forms, showing the presence of topo II beta.