Germ line-specific DNA sequences are present on all five micronuclear chromosomes in Tetrahymena thermophila.

The development of the macronucleus from the zygotic micronucleus in the ciliated protozoan Tetrahymena spp. involves the elimination of specific DNA sequences (M. C. Yao and M. Gorovsky, Chromosoma 48:1-18 1974). The present study demonstrates that micronucleus-specific DNA is present on all five of the micronuclear chromosomes. Fragments of micronuclear DNA from Tetrahymena thermophila were cloned in the plasmid vector pBR322. A procedure was developed to examine the organization of the cloned sequences in micro- and macronuclear DNA without nick translating each individual probe. Twenty-three percent of randomly selected DNA sequences examined by this method were micronucleus (germ line) specific. They were all members of families of repeated sequences. Hybridization of six micronucleus-specific DNA sequences to micronuclear DNA from nullisomic strains of T. thermophila, which are lacking one or more pairs of chromosomes in the micronucleus, suggested that these sequences are present on several chromosomes. One micronucleus-specific sequence was shown by in situ hybridization to be present on all five of the micronuclear chromosomes.

Methylation of replicating and nonreplicating DNA in the ciliate Tetrahymena thermophila.

Methylation of adenine in replicating and nonreplicating DNA of the ciliate Tetrahymena thermophila was examined. In growing cells, 87% of the methylation occurred on the newly replicated daughter strand, but methylation was also detectable on the parental strand. Methylation of nonreplicating DNA from starved cells was demonstrated.

Transformation of Tetrahymena thermophila with hypermethylated rRNA genes.

The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N6-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5'-NAT-3', the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations.

Site-specific methylation of adenine in the nuclear genome of a eucaryote, Tetrahymena thermophila.

DNA in the polyploid macronucleus of the ciliated protozoan Tetrahymena thermophila contains the modified base N6-methyladenine. We identified two GATC sites which are methylated in most or all of the 45 copies of the macronuclear genome. One site is 2 kilobases 5' to the histone H4-I gene, and the other is 5 kilobases 3' to the 73-kilodalton heat shock protein gene. These sites are de novo methylated between 10 and 16 h after initiation of conjugation, during macronuclear anlage development. The methylation states of these two GATC sites and four other unmethylated GATC sites do not change in the DNA of cells cultured under conditions which change the activity of the genes, including logarithmic growth, starvation, and heat shock.

Molecular analysis of N6-methyladenine patterns in Tetrahymena thermophila nuclear DNA.

We have cloned two DNA fragments containing 5'-GATC-3' sites at which the adenine is methylated in the macronucleus of the ciliate Tetrahymena thermophila. Using these cloned fragments as molecular probes, we analyzed the maintenance of methylation patterns at two partially and two uniformly methylated sites. Our results suggest that a semiconservative copying model for maintenance of methylation is not sufficient to account for the methylation patterns we found during somatic growth of Tetrahymena. Although we detected hemimethylated molecules in macronuclear DNA, they were present in both replicating and nonreplicating DNA. In addition, we observed that a complex methylation pattern including partially methylated sites was maintained during vegetative growth. This required the activity of a methylase capable of recognizing and modifying sites specified by something other than hemimethylation. We suggest that a eucaryotic maintenance methylase may be capable of discriminating between potential methylation sites to ensure the inheritance of methylation patterns.

A small family of elements with long inverted repeats is located near sites of developmentally regulated DNA rearrangement in Tetrahymena thermophila.

Extensive DNA rearrangement occurs during the development of the somatic macronucleus from the germ line micronucleus in ciliated protozoans. The micronuclear junctions and the macronuclear product of a developmentally regulated DNA rearrangement in Tetrahymena thermophila, Tlr1, have been cloned. The intrachromosomal rearrangement joins sequences that are separated by more than 13 kb in the micronucleus with the elimination of moderately repeated micronucleus-specific DNA sequences. There is a long, 825-bp, inverted repeat near the micronuclear junctions. The inverted repeat contains two different 19-bp tandem repeats. The 19-bp repeats are associated with each other and with DNA rearrangements at seven locations in the micronuclear genome. Southern blot analysis is consistent with the occurrence of the 19-bp repeats within pairs of larger repeated sequences. Another family member was isolated. The 19-mers in that clone are also in close proximity to a rearrangement junction. We propose that the 19-mers define a small family of developmentally regulated DNA rearrangements having elements with long inverted repeats near the junction sites. We discuss the possibility that transposable elements evolve by capture of molecular machinery required for essential cellular functions.

A developmentally regulated deletion element with long terminal repeats has cis-acting sequences in the flanking DNA.

Approximately 6000 specific DNA deletion events occur during development of the somatic macro-nucleus of the ciliate Tetrahymena. The eliminated Tlr1 element is 13 kb or more in length and has an 825 bp inverted repeat near the rearrangement junctions. A functional analysis of the cis -acting sequences required for Tlr1 rearrangement was performed. A construct consisting of the entire inverted repeat and several hundred base pairs of flanking DNA on each side was rearranged accurately in vivo and displayed junctional variability similar to the chromosomal Tlr1 rearrangement. Thus, 11 kb or more of internal element DNA is not required in cis for DNA rearrangement. A second construct with only 51 bp of Tetra-hymena DNA flanking the right junction underwent aberrant rearrangement. Thus, a signal for determination of the Tlr1 junction is located in the flanking DNA, 51 bp or more from the right junction. Within the Tlr1 inverted repeat are 19 bp tandem repeats. A construct with the 19mer repeat region deleted from the right half of the inverted repeat utilized normal rearrangement junctions. Thus, despite its transposon-like structure, Tlr1 is similar to other DNA rearrangements in Tetrahymena in possessing cis -acting sequences outside the deleted DNA.

A family of developmentally excised DNA elements in Tetrahymena is under selective pressure to maintain an open reading frame encoding an integrase-like protein.

Tlr1 is a member of a family of approximately 20-30 DNA elements that undergo developmentally regulated excision during formation of the macronucleus in the ciliated protozoan TETRAHYMENA: Analysis of sequence internal to the right boundary of Tlr1 revealed the presence of a 2 kb open reading frame (ORF) encoding a deduced protein with similarity to retrotransposon integrases. The ORFs of five unique clones were sequenced. The ORFs have 98% sequence conservation and align without frameshifts, although one has an additional trinucleotide at codon 561. Nucleotide changes among the five clones are highly non-random with respect to the position in the codon and 93% of the nucleotide changes among the five clones encode identical or similar amino acids, suggesting that the ORF has evolved under selective pressure to preserve a functional protein. Nineteen T/C transitions in T/CAA and T/CAG codons suggest selection has occurred in the context of the TETRAHYMENA: genome, where TAA and TAG encode Gln. Similarities between the ORF and those encoding retrotransposon integrases suggest that the Tlr family of elements may encode a polynucleotide transferase. Possible roles for the protein in transposition of the elements within the micronuclear genome and/or their developmentally regulated excision from the macronucleus are discussed.

Two-dimensional DNA gel electrophoresis as a method for analysis of eukaryotic genome structure: evaluation using Tetrahymena thermophila DNA.

There is growing interest in mapping and analyzing complete eukaryotic genomes. Yee and Inouye (in Experimental Manipulation of Gene Expression, pp. 279-290, Academic Press, New York) demonstrated that bacterial chromosomes can be resolved into interpretable patterns of DNA fragments by means of restriction enzyme digestion and electrophoresis in two dimensions. We have begun to explore applications of this procedure to analysis of eukaryotic genomes, which are far more complex. Tetrahymena thermophila was selected as a model organism because its genome is small, roughly equivalent to that of a single human chromosome. In addition, each Tetrahymena cell contains two nuclei which differ in sequence composition and methylation. Our results demonstrate that the Tetrahymena genome can be resolved into complex patterns of fragments in two dimensions. Hybridization to Southern blots of these gels with a multiply repeated sequence probe yielded analyzable patterns of a subset of the genome. The blots reveal alterations in genome structure due to methylation and rearrangement. Future extensions of the method are discussed.

Position effect for adenine methylation in the macronuclear DNA of Tetrahymena.

The macronucleus of the ciliate Tetrahymena contains about 45 copies of the genome. The fraction of the molecules on which an adenine residue is modified to N6-methyladenine is characteristic of the specific site, and consistent between clones. A fragment of DNA containing a site that is uniformly methylated on the macronuclear chromosome was moved to a new location on the extrachromosomal rDNA. The methylation pattern of the fragment on the rDNA was different from that on the chromosome. The data suggest that DNA sequence is not sufficient to determine the level of methylation.

The importance of surgical and multimodality treatment for small cell bronchial carcinoma.

In a cooperative international lung cancer multimodality treatment trial, 112 patients with small cell lung cancer underwent initial surgical resection and were then randomized to receive one of two intensive postoperative chemotherapeutic regimens, followed by prophylactic cranial irradiation in the disease-free patients. Regimen A consisted of eight courses of cyclophosphamide, doxorubicin, and vincristine and regimen B of two courses of three sequential drug combinations: (1) cyclophosphamide, lomustine, and methotrexate; (2) cyclophosphamide, doxorubicin, and vincristine; and (3) ifosfamid and etoposide. In 47 patients the diagnosis was known preoperatively and in 65 it was not confirmed until the resected specimen was examined (all diagnoses were reviewed by a referee pathologist). Each patient was classified by the pathologic TNM characteristics. There were 38 patients with stage I disease, 39 patients with stage II, and 35 patients with stage IIIa disease. In stage IIIa there were nine patients with T3 N0-1 disease and 26 with T1-3 N2 disease (most N2 disease was clinically undetected until thoracotomy or was discovered only by routine histologic examination of the resected mediastinal nodes). Early survival rates at 24 months calculated by the life table method are as follows: stage I, 76%; stage II, 56%; and stage IIIa, 49% (T3 N0-1, 89%; T1-3 N2, 35%). Survival rates at 36 months are 62%, 50%, and 41% (74% and 29%), respectively. The projected 36-month survival rate for 43 patients with N0 disease is 65%; for 43 with N1 disease, 52%; and for 26 with N2 disease, 29%. No difference in survival has been noted in either chemotherapy treatment group. It is concluded that initial surgical resection for limited small cell cancer (stage I, II, and T3 N0-1) followed by intensive chemotherapy is an appropriate therapeutic approach. For T1-3 N2 disease the results are inconclusive.

The role of chemotherapy including ifosfamide for ovarian carcinoma. Austrian Collab. Ovarian Cancer Study Group.

On the basis of the results of earlier studies, 30 departments of gynecology have been cooperating nationwide in Austria since 1980 to promote the use of adjuvant chemotherapy after surgery for cure of ovarian carcinoma in early stages and the role of lymph node dissection and of second-look operation. Results recorded in more than 160 patients treated with adjuvant chemotherapy after so-called radical surgery performed in disease stages I and II demonstrate that only highly differentiated tumours in stage Ia can be cured by surgery only with no further adjuvant treatment. This underlines the necessity for staging. More than 200 patients with TNM stages III and IV were randomized after debulking surgery to receive treatment with different kinds of drug combinations to compare the therapeutic efficacy of a sequential alternating drug regimen consisting of Adriamycin-cisplatin + vincristine-cyclophosphamide + high-dose methotrexate with that of the combination of Adriamycin-cyclophosphamide and that of Adriamycin-cisplatin. High-dosed ifosfamide was also used in pilot studies.

Surgery in SCLC as first step of multimodality treatment.

186 patients received surgery for cure followed by aggressive randomized chemotherapy and PCI. After a median follow-up time of about 60 months the observed 4-year overall survival rate for patients completely resected was: 58% for 68 patients with pT1-3N0M0, and 33% for 27 patients with pT1-3 pN2M0 stages of SCLC. These promising results have to be confirmed by larger cooperative studies to support the recommendation to initiate surgery without delay, i.e. without preoperative chemotherapy for SCLC as well as for the non-SCLCs. From 148 patients in ISC study III after preoperative (neoadjuvant) chemotherapy only 12 received surgery for pT1-3N2M0 but only one of them survived 36 months and none 48 months p.o.

Multimodality treatment for small cell bronchial carcinoma. Preliminary results of a prospective, multicenter trial. The ISC-Lung Cancer Study Group.

Preliminary results of the 1984 ISC (International Society of Chemotherapy) lung cancer studies I and II as of June 1990 are based on 146 patients with small cell bronchial carcinoma from 23 departments of thoracic surgery. All patients received surgery for cure in cTNM stages I and II followed by randomization for two different types of chemotherapy. For disease-free patients after completion of postoperative chemotherapy, prophylactic cranial irradiation (PCI) was administered. For the two different chemotherapeutic regimens, no statistically significant differences in survival (SVR) could be observed. Each patient was classified by the pTNM system. There were 63 patients with stage I, 44 patients with stage II and 38 patients with stage III disease. Four years after surgery, 63 patients with N0 disease had a SVR of 50%, 51 patients with N1 disease 31%, and 32 patients with N2 disease, 23%. No prolongation of brain-metastasis-free time for 62 patients receiving PCI was shown. It is concluded that initial surgical resection for small cell lung cancer in stages I and II followed by intensive chemotherapy is an appropriate therapeutic approach.

Rationale for surgery as the first step in the multimodality treatment of small cell lung cancer (SCLC).

The role of surgery has to be reconsidered in combination with chemo/radiotherapy for patients with SCLC at early stages, determined by the pTNM-staging system. This combined multimodality treatment can be seen as a model-like example for the most effective progress gained during the last 20 years. Surgery followed by chemotherapy is used in ongoing cooperative studies. In other trials, chemotherapy is followed by surgery for responding patients. It seems reasonable to expect that after appropriate observation times the comparison of these ongoing trials would most probably lead to the conclusion that for stage I, II surgery should be the first step followed by chemo- and radiotherapy, whereas for stage III debulking chemotherapy should be given to distinguish responding patients, who may thereafter receive adjuvant surgery.

Combination of surgery and chemotherapy for small-cell bronchial carcinoma.

In several test model systems using spontaneous metastasizing experimental tumours, convincing data indicate the importance of the tumour burden left after surgery for the efficacy of the combination of surgery and chemotherapy. Early removal of the primary tumour by radical surgery for cure seems to improve the conditions for chemotherapy. Since 1979, in nine different departments of thoracic surgery, patients with small-cell carcinoma of the lung (SCCL) have been randomized after surgery for cure to receive a new sequential intermittent polychemotherapy (sq.CT) of 3 different alternating drug combinations given intermittently over 1 year, or one 4-drug combination chemotherapy (CT) given intermittently over 3 years. The calculation of their life table curves at 1 August 1984 indicated an improvement in the 4-year survival rate of 23 patients receiving sq.CT to about 50%, compared with a survival rate of about 30% for 29 patients receiving CT. The number of patients is still too small for firm conclusions to be drawn, but it is concluded that surgery for SCCL seems to be an advisable measure for the efficacy of aggressive intermittent long-term polychemotherapy. However, this can only be proved in large cooperative studies.

Multimodality treatment with surgery in small-cell lung cancer.

The paper reviews the role of surgery in the local treatment of the primary tumour of small-cell bronchial carcinomas (SCLC) and the preliminary results of a multicentre cooperative prospective randomized trial with multimodality treatments of SCLC. This randomized cooperative trial was activated in 1979 comparing a new sequential intermittent polychemotherapy (sq CT) of three different drug combinations alternatingly given intermittently within one year after surgery, with a standard chemotherapy (CT) of one combination of four drugs given intermittently within three years after surgery. Observations on 25 patients at nine different cooperating surgical departments showed that sq CT was applicable and unexpected side-effects did not occur. Live table curves, calculated from 56 patients up to February 1986, indicate the improvement of the survival rate five years after surgery for the cure of 25 patients receiving sq CT after surgery in comparison with 31 patients receiving standard chemotherapy (36% : 22%). Following discussions of this and other results at the symposium "Adjuvant Therapy of Small-Cell Bronchial Carcinoma" at the 13th Congress of the International Society of Chemotherapy (ISC) 1983 in Vienna, a related new protocol was designed and activated by a further enlarged ISC-Study-Group in October 1984. This protocol for a two-armed randomized cooperative trial for patients operated for T1, 2N0M0SCLC is comparing a chemotherapy-combination 1 (CAV)--as also used in a trial of the North American Lung Cancer Study Group-0--with chemotherapy 2 (sq CT) as used in our forementioned previous study.

The importance of surgery as the first step in multimodality treatment of small cell bronchial carcinoma. The ISC Lung Cancer Study Group.

For patients with small cell lung cancer (SCLC) in their early stages (TNM I, II), surgery for cure was used to eliminate the primary tumour and its regional lymph-nodes followed by intermittent chemotherapy and radiotherapy within the first six postoperative months. After the pathohistological examination of the operation-specimen a two-arm-randomization was performed: standard chemotherapy (1000 mg/m2 cyclophosphamide, 50 mg/m2 doxorubicin, 1.4 mg/m2 vincristine) compared with sequential chemotherapy using three different drug-combinations (A: 1500 mg/m2 cyclophosphamide, 100 mg/m2 lomustine, 15 mg/m2 methotrexate; B: 1000 mg/m2 cyclophosphamide, 40 mg/m2 doxorubicin, 1 mg/m2 vincristine; C: 5 x 1.6 g/m2 ifosfamide plus mesna, 5 x 120 mg/m2 etopside). Thereafter disease-free patients only received prophylactic cranial irradiation (PCI: administering 3600 TD Gy/18 fractions) according to the protocols of the International Society of Chemotherapy Studies I and II. Preliminary evaluations in March 1990 of 170 patients from 24 cooperating departments for thoracic surgery showed that the projected life-table four-year-survival rate of 63 patients with SCLC at pTNM-stage I was 61%, of 54 patients at pTNM-stage II was 35%, of 13 patients at stage pT3, 4 NO, 1 MO was 59% and of 40 patients at stage pT N2 MO was 35%. The indication for surgery is emphasized for pTNM-stages I + II. For N2-lesions surgery would not be recommended in general, but the survival rate seems to indicate that this treatment was not detrimental, being rather more favourable compared with chemotherapy or radiotherapy alone. The continuation and enlargement of these studies seem not only justified, but emphatically indicated.(ABSTRACT TRUNCATED AT 250 WORDS)

Surgery for cure followed by combined modality treatment for small cell bronchial carcinoma. ISC Lung Cancer Study Group.

For patients with small cell lung cancer (SCLC) at early stages (TNM I, II) surgery for cure is used to eliminate the primary tumour and its regional lymph-nodes followed by intermittent chemotherapy and radiotherapy within the first six postoperative months. After the pathohistological examination of the operation-specimen a two-arm-randomization is performed: standard chemotherapy compared with sequential chemotherapy using three different drug-combinations. Thereafter tumour-free patients only receive prophylactic cranial irradiation. In preliminary evaluations of March 1988, of 121 patients from 20 cooperating departments it was found that the projected life-table survival rate, three years postoperatively, of 47 patients with SCLC at stages pT1-3 N0 M0 was 65%, of 46 patients at stages pT1-3 N1 M0, 56% and of 28 patients at stages pT1-3 N2 M0, 34%. The indication for surgery were emphasized for pTNM-stages I+II. For N2-lesions surgery would not be recommended in general, but the survival rate seemed to indicate that this treatment was not detrimental, but rather more favourable compared with chemotherapy or radiotherapy only. The continuation and enlargement of these studies seem not only justified but emphatically indicated. Multicentre cooperation has to be organized to collect within a reasonable period of time a sufficient number of patients to enable subdivisions to be made according to various prognostic factors.

Combination of surgery and polychemotherapy for cure in early small-cell bronchial carcinoma.

In 1969 a cooperative randomized two arm study of surgery and adjuvant chemotherapy was started. Within three years after the operation the polychemotherapy was given intermittently using a combination of cyclophosphamid + 5-FU + MTX + vinblastin. From a total of 859 patients operated for cure preliminary evaluations per 1.3.1981 found 53 patients with small cell type carcinomas. An increase of the 3-year survival rate of 13 per cent shows a clear positive therapeutic effect. This also demonstrates the importance of surgery even on small cell bronchogenic carcinoma by decreasing the tumor burden. To evaluate wether or not the number of long-term survivors can be increased furthermore, a new randomized trial was started in 1979 to compare the polychemotherapy used with a new designed intermittent polychemotherapy-schedule using alternating cyclophosphamid + CCNU + MTX, there after cyclophosphamid + adriamycin + vincristin and there after ifosfamid + VP-16; then radiation therapy and there after repeating the whole sequence.