Restriction Endonuclease Patterns Research Articles

Host range expansion of Autographa californica nuclear polyhedrosis virus (NPV) following recombination of a 0.6-kilobase-pair DNA fragment originating from Bombyx mori NPV

We have isolated hybrid baculoviruses of Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica NPV (AcNPV) capable of replicating in both BmN (not susceptible to AcNPV) and SF-21 (not susceptible to BmNPV) cells (A. Kondo and S. Maeda, J. Virol. 65:3625-3632, 1991). Repeated backcross infection of one of these recombinant isolates with AcNPV generated eh-AcNPV, a virus with restriction endonuclease patterns of genomic DNA nearly identical to those of AcNPV but capable of replicating in both BmN and SF-21 cells, i.e., host range expanded. Expanded host range viruses were also isolated following cotransfection of AcNPV DNA with eh-AcNPV DNA cleaved with either HindIII or PstI. Subsequent cotransfection of AcNPV DNA with plasmids from an eh-AcNPV DNA fragment library identified an 11-kbp HindIII fragment that could expand the host range of AcNPV. Subcloning and cotransfection analyses localized a 572-bp SacI-HindIII fragment within this 11-kbp fragment which could alone expand the host range of AcNPV. Mapping and nucleotide sequencing analysis revealed that this fragment was identical to the corresponding 572-bp fragment (BmScH) of BmNPV. Furthermore, this fragment originated from the coding region of the putative DNA helicase gene. Cotransfection of AcNPV DNA with BmScH also generated a host range-expanded virus, eh2-AcNPV. These results indicated that the expanded host range characteristics of eh2-AcNPV were solely the result of recombination within the coding region of the putative DNA helicase gene.

Characterization of two divergent adenovirus 31 strains

Two divergent strains of adenovirus type 31 were analyzed by neutralization test and restriction endonuclease (RE) patterns in an effort to find the basis for their genetic variability. One strain, isolated from the throat of a child in Maryland during an upper respiratory illness in 1968, was partially neutralized by Ad 31 antisera (to 16-fold lower than homologous titer) while its own antiserum fully neutralized prototype Ad 31 virus, but shared only 9% of comigrating RE fragments with Ad 31 prototype (vs. 30% with Ad 18 prototype); however, PCR tests specific for the inverted terminal repeat (ITR) sequence of Ads 12 and 18 were negative. The other strain, recovered from a stool sample from an infant with diarrhea in Georgia in 1979, was inhibited by Ad 31 antiserum to within 4-fold homologous titer, but shared only 15% of comigrating fragments with Ad 31 prototype (vs. 91% with Ad 18 prototype); ITR-specific PCR tests with this virus were positive for Ad 12/Ad 18. These data suggest that both strains are from separate evolutionary lines of Ad 31 unrelated to all other isolates studied to date by RE analysis, and that the partial neutralization by prototype Ad 31 antisera might represent small mutations in the hexon gene.

Detection of hepatitis C virus RNA in peripheral blood mononuclear cells and in saliva.

The nested polymerase chain reaction (PCR) technique was applied to investigate hepatitis C virus (HCV) RNA in the peripheral blood mononuclear cells (PBMCs), saliva, and serum of patients with chronic type C hepatitis. The specificity of the amplified products was analyzed and confirmed by agarose gel electrophoresis, Southern blot hybridization, and restriction endonuclease pattern analysis. HCV RNA was detectable in the PBMCs of 24% (12/50) of the patients. The HCV RNA detected in PBMCs was not due to the contamination from plasma, since no viral sequences could be detected in the third washing of PBMCs. Of the 12 patients with HCV RNA in PBMCs, five were negative for HCV RNA sequences in the serum. Thus the presence of HCV RNA in PBMCs was not strictly correlated to the results for sera. Among 25 patients with HCV RNA in their saliva, 18 were negative for PBMCs. Among 25 patients without HCV RNA in their saliva, five had HCV RNA in PBMCs. In conclusion, PBMCs are an extrahepatic target for chronic HCV infection. However, we do not suggest that PBMCs act as a vehicle for carrying HCV to saliva, since the presence of HCV RNA in PBMCs and in saliva was not closely correlated.

Porphyromonas endodontalis: prevalence and distribution of restriction enzyme patterns in families.

In this study we determined the prevalence and distribution of Porphyromonas endodontalis in 26 families consisting of 107 subjects. P. endodontalis was present in 24% of the investigated subjects and was recovered most often from the dorsum of the tongue (50%). Isolation was also possible from the tonsils, the buccal mucosa, the saliva and the periodontal pocket. The usefulness of restriction endonuclease analysis as a typing method for this particular species was investigated by typing 19 isolates from unrelated individuals. All these isolates had unique restriction endonuclease patterns. The observed heterogeneity indicates that restriction endonuclease analysis is a sensitive measure of genetic dissimilarity between P. endodontalis isolates and is able to characterize individual isolates. Application of restriction endonuclease analysis to the obtained clinical isolates in this study shows the possibility of the presence of multiple clonal types within one subject. The DNA patterns of all P. endodontalis isolates from unrelated individuals were found to be distinct. In 3 families the DNA patterns of isolates from the mother and her child were indistinguishable. These data indicate the possibility of intrafamilial transmission of P. endodontalis.

Genome analysis ofPropionibacterium freudenreichii by pulsed-field gel electrophoresis

Preparations of intact genomic DNA from 23 strains ofPropionibacterium freudenreichii were compared by digestion with restriction endonucleases and subsequent transverse alternating field electrophoresis (TAFE). Seven restriction enzymes,AsnI,DraI,HpaI,SnaBI,SpeI,SspI, andXbaI, produced DNA fragments useful for strain comparisons. A characteristic restriction fragment pattern was identified for 18 of the 23 strains. Estimates for the genome size of theP. freudenreichii strains ranged from 1.6×106 to 2.3×106 base pairs based on the sum of fragment sizes obtained with restriction digests. Restriction endonuclease patterns resolved by TAFE are useful for strain identification.

Characterization of newly isolated plasmids from Actinobacillus pleuropneumoniae

Summary The genetic basis of drug-resistant strains of Actinobacillus pleuropneumoniae in Japan was studied. The A pleuropneumoniae strains AV277 and AV281 that belong to serotype 2 were resistant to streptomycin (sm) and sulfonamide (sa). Both strains had an 8.1-kilobase (kb) sm-sa plasmid that was previously classified in the H1 group. The AV177 (serotype 1) strain was resistant to sm, sa, ampicillin, and kanamycin (km), but did not have any plasmids. The AV319 and AV324 (serotype 1) strains were resistant to sm, sa, tetracycline (tc), and chloramphenicol (cp). The AV318 (serotype 12) strain was resistant to sm, sa, tc, minocycline, and cp. These 3 strains (AV319, AV324, and AV318) had a 4.3-kb sm-sa plasmid and a 5.2-kb cp plasmid. The 4.3-kb plasmid was classified in the H2 group. The AV263 (serotype 1) strain was resistant to sm, sa, km, tc, and cp. It had a 5.2-kb cp plasmid and a 6.6-kb sm-sa-km plasmid. Both plasmids did not replicate stably in Escherichia coli strains. The former 5.2-kb plasmid was mobilized in E coli strains by plasmid RP4, which belonged to incompatibility P with broad host range, but the latter 6.6-kb plasmid was not so mobilized. Three 5.2-kb cp plasmids isolated from strains AV319, AV324, and AV318, had the same restriction endonuclease pattern after digestion with Ava I and EcoRI. They coexisted with H1 group plasmids in the incompatibility test, and coexisted also with H2 group plasmids of the original A pleuropneumoniae strains. Results indicated that the 5.2-kb cp plasmids could be classified in a new incompatibility group, H3. In this study, 4 types of plasmids were isolated, but no plasmids encoded tc and minocycline resistance.

Epidemiologic analysis and genotypic characterization of a nosocomial outbreak of vancomycin-resistant enterococci.

We are reporting on a nosocomial outbreak of 213 cases of vancomycin-resistant enterococcus infection involving 2,812 enterococcal isolates from patients over a period of 36 months. In 1990, the Enterococcus faecium vancomycin susceptibility rate was found to be 85.7% (36 of 42 cases), and an incidence of 10.9% (42 of 383) was noted. The 1991 data showed E. faecium with a vancomycin susceptibility rate of 61.8% (110 of 178) and an incidence of 26.0% (178 of 684). Subsequently, in 1992, the incidence of E. faecium increased to 34.0% (599 of 1,745), with a decreased vancomycin susceptibility rate of 25.8% (155 of 599). The E. faecalis vancomycin susceptibility rate remained near 97% (1,768 of 1,823) over the 36-month period. Of 115 vancomycin-resistant enterococcus (VRE) clinical isolates identified by the MicroScan MIC Combo-6 panels (Baxter Healthcare, Sacramento, Calif.), the agar dilution method indicated the resistance rate to be 92.3% (106 of 115) (high level), 3.5% (4 of 115) midlevel, and 3.5% (4 of 115) (low level). Genotypic characterization of 32 different VRE isolates by field-inversion gel electrophoresis demonstrated 19 dissimilar restriction endonuclease patterns, with 9 patterns associated with VRE quinolone resistance. Statistical analysis of case-control data for 32 patients with VRE infections indicated a positive association with intrabdominal surgical procedures (odds ratio, 24.12), multidrug therapy (odds ratio, 37.80), preexposure to vancomycin (odds ratio, 20.21), and death (odds ratio, 17.50).

Molecular analysis of highly repeated genome fractions in Solanum and their use as markers for the characterization of species and cultivars

Highly repeated DNA of potato (Solanum sp.) was characterized by cloning various major repeated elements of the nuclear genome. The percentage of the nuclear genome of the specific fractions and the restriction enzyme patterns were determined in order to show the distribution and organization of the respective repeats in the genome of Solanum tuberosum cultivars, dihaploid breeding lines and in wild species of Solanum. Several of the clones obtained were represented in a high copy number but showed no informative RFLP patterns. More information was gained from 'restriction satellite' repeats. The clone pR1T320 was found to contain satellite repeats (360 bp in length) that are proportionally present in the genome of all Solanum species at frequencies, between 0.5% and 2.6% and which are differently organized. This repeat was also found in the genera Lycopersicon, Datura and Nicotiana. With various restriction enzymes characteristic RFLP patterns were detected. A more or less genus-specific element for Solanum was the 183-bp repeat (clone pSA287; between 0.2-0.4% of the nuclear genome) that was present in the majority of the Solanum species analyzed except S. kurtzianum, S. bulbocastanum and S. pinnatisectum. In a few wild species (prominently in S. kurtzianum, S. demissum and S. acaule) a specific repeat type was detected (clone pSDT382; repeat length approximately 370 bp) that could be used to trace the wild species introduced into S. tuberosum cultivars. The repeats analyzed together with the 18S, 5.8S and 25S ribosomal DNA (1.9-5.2%, corresponding to 1800-5500 rDNA copies) comprised approximately 4-7% of the Solanum genome.

Role of gastric pH in isolation of Helicobacter mustelae from the feces of ferrets

Background: Helicobacter mustelae colonizes the gastric mucosa of ferrets and causes persistent chronic gastritis. Methods: Hypochlorhydria, as measured by gastric pH probe, was induced by administering oral omeprazole, a proton pump inhibitor of the parietal cell, to adult ferrets in two separate experiments. Feces of ferrets were cultured for H. mustelae before, during, and after omeprazole therapy. Results: H. mustelae was isolated in 23 of 55 (41.8%) sequential fecal samples collected during omeprazole therapy. The same ferrets with acidic gastric pH had H. mustelae isolated in 6 of 62 (9.7%) of the fecal cultures (P < 0.01). A DNA species-specific H. mustelae probe confirmed the presence of the organism in both the stomach and feces of all 5 ferrets. In 4 of 5 ferrets restriction enzyme patterns of the gastric H. mustelae were identical to those of the fecal H. mustelae strains. Conclusions: Hypochlorhydria promotes fecal transmission of a gastric Helicobacter organism. The H. mustelae-colonized ferret provides an ideal model to study the epidemiology and pathogenesis of Helicobacter pylori-induced gastritis.

Homologous recombination of adenovirus DNA in mammalian cells: enhanced recombination following UV-irradiation of the virus

We have used adenovirus as a molecular probe to examine the recombination of viral DNA following infection of mammalian cells. The technique gives a quantitative measure of homologous recombination between adenovirus type 2 (Ad2) and Ad5PyMTR3. Ad5PyMTR3 is an insertion mutant of Ad5 containing polyoma virus (Py) DNA inserted into a deleted E1 region of the Ad5 genome. Cells were coinfected with Ad2 and Ad5PyMTR3 and at an appropriate time after infection, viral DNA was extracted from the infected cells, digested with restriction endonuclease and electrophoresed through an agarose gel. Although Ad2 and Ad5 have more than 99% DNA homology, they differ sufficiently in their restriction endonuclease patterns, such that recombinant viral DNA molecules containing the Py insert could be detected and quantified by Southern blotting and hybridization to a radioactive Py DNA probe. Using this method we are able to detect and quantitate recombinant viral DNA molecules containing the Py insert which are present at frequencies down to at least 1 in 100. Recombination was detected in Chinese hamster ovary cells, monkey kidney cells, human HeLa cells, normal human fibroblasts and SV40 transformed human fibroblasts. In experiments using HeLa cells, the frequency of recombination between the Py insert on Ad5PyMTR3 and a number of unique restriction enzyme sites on Ad2 increased with the distance from the Py insert to the restriction site. Also in HeLa cells, recombination increased with increasing amounts of viral DNA synthesis and with increasing UV dose to the virus. UV-irradiation of both coinfecting viruses with 1500 J/m2 resulted in a more than 100-fold reduction in the amount of viral DNA synthesized and about a 3-fold increase in the frequency of recombination.

Isolation of Human Immunodeficiency Virus Type 1 from Human Epidermis: Virus Replication and Transmission Studies

For a better understanding of the pathogenetic events operative in the cutaneous manifestations of human immunodeficiency virus type 1 (HIV-1) disease, we investigated whether epidermal cells (EC) from HIV-1-seronegative persons can be infected with HIV-1 and, vice versa, whether HIV-1 can be rescued from the epidermis of HIV-1-infected individuals. In a series of three experiments, we consistently found that exposure of EC from HIV-1-seronegative donors to HIV-1 led to viral replication in these cells as evidenced by the detection of HIV-1 p24 in culture fluids. Because EC had been substantially enriched for Langerhans cells (LC) before being exposed to HIV-1, it is reasonable to assume that these CD1a+/CD4+/MHC class II+ antigen-presenting cells of the epidermis represented the actual targets of infection. This assumption is further strengthened by the observation that T cell-depleted cell suspensions from Langerhans cell histiocytosis (LCH) lesions could be productively infected with HIV-1. Conversely, co-culture of epidermal sheets from HIV-1-seropositive individuals with mononuclear phagocytes (MNP) from HIV-1-seronegative donors resulted, after 3 to 5 weeks, in the detection of HIV-1 p24 in 12 of 23 cases. Immunocytochemical analysis, using a monoclonal antibody specific for p24, revealed the presence of HIV-1 in adherent MNP in three cocultures tested. In addition, cellular DNA from these cultures showed strong signals when hybridized to a HIV-1-specific DNA probe. The further finding that two isolates examined exhibited different restriction enzyme patterns indicates that they are separate entities rather than contaminants. Transmission of these isolates to MNP, B- or T-cell lines resulted in cultures strongly positive for p24 and, in the case of H9 cells, for viral particles as detected by electron microscopy. Our results therefore strongly suggest that EC not only can serve as targets for HIV-1, but also can allow efficient virus replication and transmit HIV-1 to various cell types of the hematopoietic lineage.

Restriction endonuclease patterns of adenovirus type 12 and 18

Restriction endonuclease analysis using 10 restriction enzymes was performed on six and three wild isolates of adenovirus (Ad) type 12 and 18, respectively. Among the Ad 12 strains, five DNA variants could be identified. The degree of pairwise comigration of restriction fragments suggests a high degree of genomic diversity within Ad12. The wild isolates of Ad18, on the other hand, displayed a low degree of genetic variability and comprised one DNA variant closely related to the prototype strain. The BglII, BstEII, and HindIII restriction endonuclease patterns of Ad18 were inconsistent with those originally presented. Identical RE-patterns among Ad18 prototype strains (DC) obtained from four different sources, including directly from the American Type Culture Collection, verify that the genuine Ad18 prototype was used in the present study. Using the revised restriction patterns of BglII, BstEII, and HindIII, a proper identification of Ad18 will be facilitated.

Identification and epidemiological typing of clinical and environmental isolates of the genus Rhodococcus with use of a digoxigenin-labeled rDNA gene probe.

Rhodococcus species are ubiquitous in the environment, and several species have been reported to have pathogenic potential for humans. Rhodococcus equi, in particular, has been reported to cause infections in patients with AIDS. However, the identification of Rhodococcus species with use of conventional biochemical tests is problematic, and no simple and reproducible method exists for their rapid identification and differentiation. We found that the type strains of the 20 recognized species in the genus Rhodococcus could be clearly distinguished with use of a combination of the Pvu II and Pst I rRNA gene restriction endonuclease patterns and a digoxigenin-labeled Escherichia coli rDNA probe. Analysis of four clinical or environmental isolates confirmed as Rhodococcus bronchialis showed no interstrain variation of rRNA gene bands. Analysis of 15 isolates confirmed as R. equi from 13 patients showed 11 different rRNA gene patterns. No discernible difference was observed in the ribotype patterns between R. equi isolates from patients for whom AIDS had been diagnosed and those from patients who did not have AIDS, and there was no evidence of geographic clustering of R. equi ribotype patterns. Three of five Rhodococcus species isolates that could not be differentiated with use of conventional biochemical methods were identified with use of ribotype analysis. Therefore, ribotype analysis may provide an important adjunct to current biochemical identification of environmental and clinical isolates of Rhodococcus species.

Recovery of recombinant plasmids from Pleurotus ostreatus transformants.

A transformation system employing selectable resistance to hygromycin B has been developed for the mushroom-forming fungus, Pleurotus ostreatus. Vector pAN7-1, a commonly used non-replicative vector for integrative transformation in fungi, yielded 5-46 resistant colonies per micrograms of DNA per 10(7) viable protoplasts. Southern blot analysis of certain transformants revealed unexpected replicative plasmids containing pAN7-1 sequences, but modified for size, methylation and restriction enzyme pattern when compared to the initial transforming vector. Two such replicative derivatives of pAN7-1 have been rescued from P. ostreatus by cloning into Escherichia coli. Rescued plasmids have been used to probe DNA from untransformed P. ostreatus in an effort to identify fungal sequences that recombined in vivo with pAN7-1 to form replicative plasmids. Such replicative sequences have been localized in high molecular weight (chromosomal) DNA of wild-type P. ostreatus. Transformation has been obtained for P. ostreatus using a rescued plasmid, thereby confirming the role of this recombinant plasmid as a shuttle vector.

Conjugative gene transfer in marine cyanobacteria: Synechococcus sp., Synechocystis sp. and Pseudanabaena sp.

Versatility of gene transfer by transconjugation in marine cyanobacteria was demonstrated. In this study, seven different marine cyanobacteria were used as recipient cells. First, transconjugation was carried out using the mobilizable transposon (Tn5) carrying plasmid pSUP1021. Transconjugates were observed in all marine cyanobacteria tested. Second, the broad-host-range vector pKT0230 (IncQ) was tested for transconjugation. pKT230 has been successfully transferred in a marine cyanobacterium Synechococcus sp. NKBG15041C, and replicated as an autonomous replicon without alteration in the restriction enzyme pattern. A maximum transfer efficiency of 5.2 x 10(-4) transconjugants/recipient cell was observed, when mating was performed on agar plates containing low salinity (0.015 M NaCl) medium. This is the first study to demonstrate gene transfer in marine cyanobacteria via transconjugation.

Characterization of the Virulence Regions in the Plasmids of Three Live Salmonella Vaccines

Three live Salmonella vaccines, Zoosaloral "Dessau", Bovisaloral "Dessau", and Suisaloral "Dessau" successfully used in veterinary medicine in eastern Germany were analysed for their plasmid DNA content. Plasmids of 60 MDa (S. typhimurium), 55 MDa and 26 MDa (S. dublin) and 34 MDa (S. choleraesuis) were found. The three large plasmids contained a virulence region as shown by restriction enzyme analysis and hybridization studies with virulence-region-specific DNA probes. Restriction enzyme pattern analysis of the whole plasmids revealed differences between vaccine strains and parental strains. We wanted to know if there were alterations within the virulence region of the vaccine strain plasmids compared with parental strains caused by the attenuation procedure. Therefore, the 6 kb-Cla I fragments of several plasmids were cloned and characterized by restriction enzyme fingerprinting. The expression of the Cla I fragment-encoded proteins was analysed in the minicell-producing strain DS 410. The experiments revealed that the restriction pattern of the 6 kb-Cla I fragments of Zoosaloral and Bovisaloral as well as the PAGE pattern of virulence-region-encoded proteins were in complete accordance with one another, between vaccine strains and the parental strain of Zoosaloral, and with a wild-type strain of S. typhimurium.

Primary structure of the Aequorea victoria green-fluorescent protein

Many cnidarians utilize green-fluorescent proteins (GFPs) as energy-transfer acceptors in bioluminescence. GFPs fluoresce in vivo upon receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca 2+-activated photoprotein. These highly fluorescent proteins are unique due to the chemical nature of their chromophore, which is comprised of modified amino acid (aa) residues within the polypeptide. This report describes the cloning and sequencing of both cDNA and genomic clones of GFP from the cnidarian, Aequorea victoria. The gfp10 cDNA encodes a 238-aa-residue polypeptide with a calculated M r of 26888. Comparsion of A. victoria GFP genomic clones shows three different restriction enzyme patterns which suggests that at least three different genes are present in the A. victoria population at Friday Harbor, Washington. The gfp gene encoded by the λGFP2 genomic clone is comprised of at least three exons spread over 2.6 kb. The nucleotide sequences of the cDNA and the gene will aid in the elucidation of structure-function relationships in this unique class of proteins.

DNA fingerprinting in the epidemiology of African serogroup A Neisseria meningitidis.

The restriction endonuclease (RE) technique was used to compare 172 meningococcal group A strains collected between 1969 and 1990, mainly from countries of the so-called African Meningitis Belt, the Gambia and Ethiopia. The 64 strains from various African countries (Niger, Chad, Burkina Faso, Cameroon, Morocco, Djibouti) were distributed within 3 main restriction enzyme patterns (REPs); the 77 Gambian strains fell into 5 REPs and the 24 Ethiopian strains into 2 such patterns. Several of the main REPs were formed by clusters of closely related clones. Clones, very similar to dominating REPs of the 1960s in Niger, Burkina Faso and Cameroon, were in the 1980s found to be strongly represented in the Gambia to the extreme west of the Meningitis Belt. One of the Gambian clones from 1983-86 was identical to an Indian clone recovered in New Delhi 1986-87. Another clone was detected in 1983 in the Gambia, in 1989 again in the Gambia as well as in Ethiopia, and in 1990 in Tanzania. Our results are largely in line with those of previous studies based on modern techniques of protein and isoenzyme electrophoresis. The RE method is useful mainly for the exact genotypic differentiation of closely related clones, and seems to be a valuable complement to phenotypic tools for epidemiological mapping of Group A meningococcal infection.

Colonisation of the respiratory tract with Legionella pneumophila for 63 days before the onset of pneumonia

We report the case of a 70-year-old man who was admitted to hospital A 66 days before developing Legionella pneumophila pneumonia 6 days after open heart surgery at hospital C. The strain of L. pneumophila recovered from the patient's sputum was of the same subtype (monoclonal antibody type, enzyme type, plasmid profile, and restriction endonuclease pattern) as a strain of L. pneumophila in the potable water supplied to the room where he stayed in hospital A. We conclude that the patient's respiratory tract became colonised by L. pneumophila while he was in hospital A and persisted for at least 63 days until he developed pneumonia requiring antibiotic treatment while in hospital C.

Changes in protein synthesis without evidence of DNA methylation in barley chloroplasts during leaf growth and development

The polypeptides synthesized by isolated chloroplasts and the endonuclease restriction patterns of plastid DNA have been investigated during greening, expansion, maturity and senescence of the primary leaf of barley. Greening chloroplasts synthesized diverse polypeptides, among which those presumably corresponding to large subunit of rubisco (54 kDa) and photosystem II D1 protein (32 kDa). During leaf expansion and maturation the activity of protein synthesis decreased and the electrophoretic profile of the polypeptides synthesized changed. The pattern of the restriction fragments produced by digestion with EcoRI, HaeIII, HpaII and MspI was indistinguishable among plastid DNAs of leaves at different stages of development. Moreover, there was no difference between the restriction patterns produced by the isoschizomers HpaII and MspI which differ in their sensitivity to methylation. Apparently, no plastid DNA rearrangement, gain, loss or methylation causes the decrease in mRNAs and protein synthesis and the change in polypeptides synthesized by chloroplasts during leaf growth and senescence.