DNA methylation profiles differ between field- andin vitro-grown leaves of apple

A reliable method has been previously developed to detect cytosine methylation at the 5′-CCGG-3′ sequence using isoschizomers (Msp I and Hpa II) and a modified amplified fragment length polymorphism (AFLP) technique. With this method, DNA methylation profiles were investigated in leaf tissues of apple (Malus × domestica cv. Gala) grown under two different growth conditions, field and tissue culture. A total of 1,622 AFLP bands were detected using 32 pairs of primers, and these banding patterns were assembled into three groups. Type I AFLP bands were present in both EcoR I/Hpa II and EcoR I/Msp I lanes. Type II bands were present in the EcoR I/Msp I lanes, but not in EcoR I/Hpa II lanes. Type III bands were present in EcoR I/Hpa II lanes, but not in the EcoR I/Msp I lanes. For leaf tissues of field- and in vitro-grown apples, the ratios of types I, II, and III to the total number of amplified fragments were 70 %, 24 %, and 6 %, and 71 %, 23 %, and 6 %, respectively. Although the ratios of the three types of banding patterns were similar in both leaf tissues, a few bands specific to either field-grown trees or in vitro-grown shoots were observed. This study provided evidence that changes in methylation occurred in apple leaf tissues subjected to tissue culture growth conditions.     

Evaluation of Genetic and Epigenetic Modification in Rapeseed （Brassica napus） Induced by Salt Stress

Salinity is an important limiting environmental factor for rapeseed production worldwide. In this study, we assessed the extent and pattern of DNA damages caused by salt stress in rapeseed plants. Amplified fragment length polymorphism （AFLP） analysis revealed dose-related increases in sequence alterations in plantlets exposed to 10-1000 mmol/L sodium chloride. In addition, individual plantlets exposed to the same salt concentration showed different AFLP and selected region amplified polymorphism banding patterns. These observations suggested that DNA mutation in response to salt stress was random in the genome and the effect was dose-dependant. DNA methylation changes in response to salt stress were also evaluated by methylation sensitive amplified polymorphism （MSAP）. Three types of MSAP bands were recovered. Type Ⅰ bands were observed with both isoschizomers Hpa Ⅱ and Msp Ⅰ, while type Ⅱ and type Ⅲ bands were observed only with Hpa Ⅱ and Msp Ⅰ, respectively. Extensive changes in types of MSAP bands after NaCI treatments were observed, including appearance and disappearance of type Ⅰ, Ⅱ and Ⅲ bands, as well as exchanges between either type Ⅰand type Ⅱ or type Ⅰ and type Ⅲ bands. An increase of 0.2-17.6% cytosine methylated CCGG sites were detected in plantlets exposed to 10- 200 mmol/L salt compared to the control, and these changes included both de novo methylation and demethylation events. Nine methylation related fragments were also recovered and sequenced, and one sharing a high sequence homology with the ethylene responsive element binding factor was identified. These results demonstrated clear DNA genetic and epigenetic alterations in planUets as a response to salt stress, and these changes may suggest a mechanism for plants adaptation under salt stress.     

DNA-methylation alterations and exchanges during in vitro cellular differentiation in rose (<Emphasis Type="Italic">Rosa hybrida</Emphasis> L.)

DNA-methylation profiles of leaf tissues of Rosa hybrida cv. Carefree Beauty collected from in vivo-grown greenhouse plants, in vitro-grown proliferating shoots at different passages, regenerants of embryogenic callus, regenerants of organogenic callus, as well as calli from undifferentiated callus (UC), embryogenic callus, and organogenic callus were investigated using an amplified fragment-length polymorphism (AFLP)-based detection technique. Three types of AFLP bands were recovered. Type I bands were observed with both isoschizomers Msp and HpaII, while type II and type III bands were observed only with MspI and HpaII, respectively. Sequence analysis of the three types of AFLP bands revealed that a nonmethylated MspI/HpaII-recognition site 5-CCGG-3 resulted in a type I band, while an inner 5-methylcytosine generated most type II and type III bands. About 40% of inner and 20% of outer cytosines in 5-CCGG-3 sequences were fully methylated, and only a few hemimethylated outer cytosines were observed. Changes in types of AFLP bands among different tissues were frequently observed, including appearance and disappearance of type I, II, and III AFLP bands, as well as exchanges between either type I and type II or type I and type III AFLP bands. Methylation alterations of outer cytosines in 5-CCGG-3 sequences triggered appearance and disappearance of type I and II AFLP bands. Methylation changes of both outer and inner cytosines resulted in either removal or generation of type III AFLP bands. Methylation alteration of an inner cytosine was responsible for exchange between type I and type II, while hemimethylation of an outer cytosine accounted for exchange between type I and type III AFLP bands. During UC induction, a significant DNA-methylation alteration was detected in both inner and outer cytosines. Variations in methylation profiles significantly differed between somatic embryogenesis and in vitro organogenesis. Demethylation of outer cytosines occurred at a high frequency during somatic embryogenesis, and most altered AFLP bands in embryogenic callus were passed on to its regenerants. However, most methylation-altered AFLP bands during organogenesis were recovered in shoot regenerants derived via organogenic callus. Seven tissue-specific bands were isolated, cloned, and sequenced. Blast search revealed that two of these might be derived from functional genes.Mingliang Xu and Xiangqian Li contributed equally to this paper     

Growth conditions of F9 embryonal carcinoma cells affect the degree of DNA methylation

We have investigated differences in C^*pG methylation between F9 embryonal carcinoma cellsin vitro and as tumor cells grownin vivo usingMsp I andHpa II restriction isoschizomers. Southerns were hybridized with two low copy number probes, mouse major -globin (f7) and a class I, histocompatibility-2 cDNA clone (pH-2^d-4). In each case, the tumor-DNA was hypomethylated while the DNA from F9 cells grownin vitro was moderately methylated. We conclude that growth conditions or cell-cell interactions can greatly affect methylation of C^*pG sites.     

Variation of cytosine methylation in 57 sweet orange cultivars

Sweet orange is an important group of citrus cultivars, which includes a number of bud sport cultivars. Little is known about the CpG methylation status of the CCGG sequences in the orange genome. In this study, methylation-sensitive amplification polymorphism (MSAP), based on the application of isoschizomers (Hpa II and Msp I), was first used to analyze cytosine methylation patterns in 57 orange cultivars that were not fully differentiated by regular DNA molecular markers. Three types of bands were generated from ten primer pairs. Type I bands were present following restriction with Eco RI + Hpa II and Eco RI + Msp I; type II or type III were present only following restriction with either Eco RI + Hpa II or with Eco RI + Msp I. The total number of these three types of bands was 802, 72, and 157, respectively. Among these, the number of polymorphic bands were 244 (30.2%), 23 (31.9%), and 32 (20.4%), in type I, II and III, respectively. The methylation patterns of these 57 cultivars are discussed and assessed by dendrograms derived from the analysis of polymorphic MSAP bands. The distribution of polymorphic bands of the above three types demonstrate the methylation patterns and frequency at the cytosine loci. We suggest that methylation events could be more frequent than demethylation events, and that the methylation patterns maybe associated with phenotypic traits.     

Clusters of repeated sequences of chicken DNA are extensively methylated but contain specific undermethylated regions

In the chicken genome there are middle repetitive DNA sequences with a clustered organization. Each cluster is composed of members of different families of repeated DNA sequences and usually contains only one member of each family. Many clusters have the same assortment of repeated sequences but they are in scrambled order from cluster to cluster. These clusters usually exceed 20 × 10³ bases in length and comprise at least 10% of the repeated DNA of the chicken. The repeated sequences that are cluster components are extensively methylated. Methylation was detected by comparing HpaII and MspI digests of total DNA, where the occurrence of the sequence C-m⁵C-G-G is indicated when HpaII (cleaves C-C-G-G) fragments are larger than those generated by MspI (cleaves C-m⁵C-G-G or C-C-G-G). In hybridization experiments with Southern (1975) blots of total DNA digested with either HpaII or MspI, the cloned probes representing clustered repeated sequences showed a dramatic difference in the lengths of restriction fragments detected in the two digests. Many of the sequences that comprise these clusters are methylated in most of their genomic occurrences. There are patterns of methylation that are reproduced faithfully from copy to copy. The overall distribution of methylation within clusters seems to be regional, with long methylated DNA segments interrupted by specific undermethylated regions.     

Gene expression,X-inactivation,and methylation during spermatogenesis: The case of Zfa,Zfx, and Zfy in mice

While it has become clear that X-inactivation in the female soma is complete in mouse (in contrast to being “patchy” in man), the degree of X-inactivation in the testes has not been ascertained. We have compared autosomal and X-linked zinc finger homolog expression and X-linked and Y-linked zinc finger homolog methylation in an attempt to elucidate this question. Using RTPCR, we have extended earlier studies of Zfx and Zfa expression in developing testes and find that Zfa expression starts at the time of X-inactivation while Zfx expression is continuous. Cell separation studies did not preclude continued expression of Zfx in adult germ cells. The methylation status of four CCGG residues in the Zfx promoter was studied using PCR bridging this region before and after DNA digestion with the isoschizomers Msp I and Hpa II, the latter being methylation sensitive. Hpa II resistant Zfx promoter DNA was found in all female tissues, but not in male tissues, including the testes. Previous studies have shown that Zfy is expressed at meiosis (like Zfa and unlike Zfx). Despite its expression, the Zfy gene is adjacent to, or contains, highly methylated CCGG sites since hybridization after Msp I digestion detected multiple small fragments that were not released after DNA digestion with Hpa II. Thus, Zfx is not methylated in sperm, while Zfy is, in contrast to their apparent patterns of expression. © 1993 Wiley-Liss, Inc.     

Analysis of DNA methylation patterns of PLBs derived from <Emphasis Type="Italic">Cymbidium hybridium</Emphasis> based on MSAP

The best known and most thoroughly studied epigenetic phenomenon is DNA methylation, which plays an important role in regulating gene expression during plant regeneration and development. In this study, the methylation-sensitive amplified polymorphism (MSAP) technique was carried out to determine differences in methylation profiles between two forms of protocorm-like bodies (PLBs), continuously proliferating PLBs (cPLBs) and spontaneously-differenting PLBs (sdPLBs), derived from cultures of Cymbidium hybridium. A total of 72 selective primer combinations were used to assess the status of cytosine methylation of DNA in these tissues. Of 4,440 fragments obtained 911 fragments, each representing a recognition site cleaved by one or both of the isoschizomers (Hpa II and Msp I), were amplified and were significantly different between the two forms of PLBs. Frequency of total and full-methylation of cPLBs and sdPLBs were 26.7/12.2%, 24.1/11.1%, respectively. In addition, 14 types of MSAP patterns detected in the two forms of PLBs belonged to two classes, type I and II. Sequencing of 14 differentially methylated fragments and their subsequent blast search revealed that cytosine methylated 5′-CCGG-3′ sequences were equally distributed in the coding and non-coding regions. Southern blotting was conducted to verify the methylation polymorphism.     

CpG islands detected by self-primed in situ labeling (SPRINS)

We have studied the distribution and methylation of CpG islands on human chromosomes, using the novel technique of self-primed in situ labeling (SPRINS). The SPRINS technique is a hybrid of the two techniques primed in situ labeling (PRINS) and nick translation in situ. SPRINS detects chromosomal DNA breaks, as in nick translation in situ, and not annealed primers, as is the case in PRINS. We analyzed in situ-generated DNA breaks induced by the restriction enzymes HpaII and MspI. These restriction enzymes enable the detection of chromosomal CpG islands. Both HpaII- and MspI-SPRINS produce a banding pattern resembling R-banding, indicating a higher level of CpG islands in R-positive bands than in R-negative bands. Our SPRINS banding observations also indicate differences in sequence copy number in the satellites of homologous acrocentric chromosomes. Furthermore, a comparison of homologous HpaII-SPRINS-banded X chromosomes of females from lymphocyte cultures grown without methotrexate or bromodeoxyuridine revealed methylation difference between them. The same comparison of homologous X chromosomes from the cell line GM01202D, which has four X chromosomes, one active and three inactive, revealed the active X chromosome to be hypermethylated. Received: 5 February 1998; in revised form: 8 May 1998 / Accepted: 11 May 1998     

Construction of Libraries for Methylation Sites by In-gel Competitive Reassociation (IGCR)

The in-gel competitive reassociation (IGCR) procedure was successfullyapplied to construct a comprehensive library enriched in DNAfragments containing C^5mCGG sequences from mouse liver and braingenomic DNA. For IGCR, methylation-insensitive restriction enzyme(Msp I) digests were used as target DNA and methylation-sensitiverestriction enzyme (Hpa II) digests as competitor DNA. Southernblot analysis indicated that 60 to 70% of the clones in thelibrary were derived from the methylated sites and overall enrichmentwas 200- to 1000-fold. IGCR was further applied to constructa library for the sites differentially methylated between brainand liver DNA. In the library, approximately 20% of the HpaII sites exhibited different degrees of methylation betweenthese tissues.     

Methylation of mitochondrial DNA in carrot (Daucus carota L.)

The methylation status of carrot (Daucus carota L.) mitochondrial DNA (mtDNA) was studied using isoschizomeric restriction enzymes MspI/HpaII (CCGG) and MvaI/EcoRII [CC(A/T)GG]. Southern hybridisations with probes for mitochondrial genes coxII and atpA were performed. MtDNAs isolated from non-embryogenic cell suspensions and roots were analysed. No differences were found using MspI/HpaII but after digesting the mtDNA with MvaI and EcoRII, some qualitative and quantitative differences between the restriction patterns appeared. Distinction was also revealed after Southern hybridisation with the coxII probe. These data indicate that the mtDNA of carrot is methylated in CNG trinucleotides and unmethylated in CG dinucleotides in CCGG sequences. The results were reproducible for cell suspensions of various genotypes and even cultivars but the extent of methylation was different in the root. The possible role of methylation in the mitochondrial genome of higher plants is discussed. Received: 16 April 1997 / Revision received: 4 July 1997 / Accepted: 30 July 1997     

DNA Methylation Occurred around Lowly Expressed Genes of Plastid DNA during Tomato Fruit Development

We have analyzed DNA methylation of plastid DNA from fully ripened red fruits, green mature fruits, and green leaves of tomato (Lycopersicon esculentum var. Firstmore). Essentially identical restriction profiles were obtained between chromoplast and chloroplast DNAs by EcoRI digestion. BstNI/EcoRII and HpaII/MspI are pairs of isoschizomers that can discriminate between methylated and unmethylated DNAs. These endonucleases produced different restriction patterns of plastid DNAs from tomato fruits compared to tomato leaves. Moreover, we have found from Southern blots that methylation was not detected in DNA fragments containing certain genes that are actively expressed in chromoplasts, whereas DNA fragments bearing genes that are barely transcribed in chromoplasts are methylated.     

Characterization of DNA methylation in the rat

In the rat, differentiation and cell proliferation both affect DNA methylation. We studied 5-methylcytosine at the inner cytosine of the sequence C-C-G-G, a common methylation site, using endonuclease MspI (which cleaves C-C-G-G- and C-^mC-G-G), and its isoschizomer HpaII (which cleaves only C-C-G-G). DNA from all tissues and cell lines studied was methylated at C-C-G-G, at levels ranging from 45 to 80%, but the methylation sites were not distributed uniformly. Our analysis suggests a model in which cells contain variable amounts of three DNA methylation states, averaging 30–40, 70–80 and 95–100% methylation, respectively. One biological parameter that alters methylation is the prolferative state of the cell. We observed that NRK, a non-transformed cell line, increased its DNA methylation from 45 to 67% when monolayer cultures became confluent and non-dividing. We also observed that a class of repetitive DNA was completely methylated in DNA from all sources except a transformed cell line.     

Hypermethylation of tobacco heterochromatic loci in response to osmotic stress

 Plants have to cope with a number of envi-ronmental stresses which may potentially induce genetic and epigenetic changes and thus contribute to genome variability. In the present study we inspected the DNA methylation status of two heterochromatic loci (defined with repetitive DNA sequences HRS60 and GRS) in a tobacco cell culture exposed to osmotic stress. Investigations were performed on a TBY-2 cell suspension culture, and the stress was elicited with NaCl or D-mannitol. Using the restriction enzymes MspI/HpaII and MboI/Sau3AI in combination with Southern hydridization we observed a reversible hypermethylation of the external cytosine at the CpCpG trinucleotides in cells grown under mild osmotic stress equal to a NaCl concentration of 10 g/l. There were no changes in the methylation of the internal cytosine as the CpG dinucleotides within the CCGG motifs (HpaII sites) appeared to be fully methylated in tobacco DNA repetitive sequences under normal physiological conditions. The data suggest epigenetic changes in the plant genome based on de novo methylation of DNA in response to environmental stress. Received: 26 November 1996/Accepted: 20 December 1996     

Methylation and expression of a metallothionein promoter ovine growth hormone fusion gene (MToGH1) in transgenic mice

We have examined transgene methylation in the DNA from the livers of a pedigree of mice carrying three copies of an integrated MToGH1 transgene. Utilizing the methylation-sensitive isoschizomersMsp I andHpa II, Southern blot analysis revealed that all second generation animals derived from a transgenic female had hypermethylated DNA, whereas first generation animals sired by a transgenic male displayed a range of methylation phenotypes ranging from no methylation to hypermethylation of the transgene sequences. Of the mice that exhibited hypermethylation of the transgene in CpG dinucleotides (CmCGG), a minority of these animals also exhibited apparent CpC methylation (i.e. inhibition ofMsp I cutting, presumably blocked by methylation of the outer C of CCGG). Methylation was also examined in the inner C of CC(A/T)GG sequences in the MToGH1 transgene using the isoschizomer pairBstN I andEcoR II. A minority of MToGH1 animals in the F₁ generation showed clear evidence of methylation in these sites as well as in the inner and outer Cs of CCGG sites. An examination of MToGH1 expression in terms of oGH levels in serum revealed that there was a high degree of variation in the levels of circulating oGH between animals of this pedigree. There was a weak inverse relationship between the serum level of oGH and the extent of methylation of the transgene. In particular, mice exhibiting CpC together with CpG methylation were found to have very low levels of circulating oGH. Our results highlight the nature and complexity of epigenetic factors associated with transgene sequences which may ultimately influence expression of introduced genes in the mammalian genome.     

Tissue culture-induced locus-specific alteration in DNA methylation and its correlation with genetic variation in <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> Benth. et Hook. f

We have reported recently that tissue culture induced a high level of genetic variation at the primary nucleotide sequence in regenerants of medicinal plant Codonopsis lanceolata. It is not known, however, whether epigenetic variation in the form of alteration in DNA methylation also occurred in these plants. Here, we investigated possible alterations in level and pattern of cytosine methylation at the CCGG sites in the same set of regenerants relative to the donor plant, by the MSAP method employing a pair of isoschizomers, HpaII and MspI, which recognize the same restriction site but are differentially sensitive to cytosine methylation at the CCGG sites. A total of 1,674 MSAP profiles were resolved using 39 primer combinations. Of these, 177 (10.5%) profiles were polymorphic among the regenerants and/or between the regenerant(s) and the donor plant, in EcoRI + HpaII or EcoRI + MspI digest but not in both, indicating alteration in cytosine methylation patterns of specific loci, though their estimated total level of methylation remained more or less the same as the donor plant. Gel blot analysis validated most of the variant MSAP profiles as bona fide alteration in methylation patterns. Correlation analysis between the MSAP data and the previously reported ISSR and RAPD data revealed significant correlations, suggesting their possible intrinsic interrelatedness. Thirty-seven typical variant MSAP profiles were isolated and sequenced, of which 5 showed significant homology to known-function genes, 2 to chloroplast sequences, whilst the rest 30 did not find a match in the database. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. W. L. Guo and R. Wu contributed equally to this work.     

DNA methylation affected by male sterile cytoplasm in rice (Oryza sativa L.)

Male sterile cytoplasm plays an important role in hybrid rice, and cytoplasmic effects are sufficiently documented. However, no reports are available on DNA methylation affected by male sterile cytoplasm in hybrid rice. We used a methylation-sensitive amplified polymorphism technique to characterize DNA methylation in four male sterile cytoplasms that are widely commercialized in China. In total, 12 pairs of selective primers in combinations of EcoRI and MspI/HpaII amplified 350 bands among four male sterile (A) lines and the corresponding maintainer (B) lines. Sites b1 and b3 were fully methylated only in all the B lines, while b2 was fully methylated only in all the A lines. These results implied a relationship of DNA methylation at these sites specifically with male sterile cytoplasms, as well as male sterility, since the only difference between the A and B lines was the cytoplasm. The DNA methylation was markedly affected by male sterile cytoplasms. WA-type and Yinshui-type cytoplasms affected the methylation to a much greater degree than G-type and D-type cytoplasms, as indicated by the number and degree of methylated sites, ratio of methylated sites, number of fully methylated sites, ratio of fully methylated sites, and polymorphism between A and B lines for these cytoplasms. The genetic distance between the cytoplasm and nucleus for the WA-type is much greater than for G- and D-types because the former is between wild and cultivated species and the latter is within indica subspecies between African and Asian cultivars. This difference in genetic distance may be responsible for the variation in methylation which we observed.     

The Genome of the Lepidopteran Mamestra brassicae has a Vertebrate-like Content of Methyl-cytosine

Mamestra brassicae genomic DNAs, isolated from larvae and adult tissues and from in vitro cultured CRL-8003 cells, were enzymatically hydrolysed to nucleosides that were separated by HPLC. HPLC analysis showed that 5mC content in cabbage moth larvae, adults and cultured cells was 8.9±0.5, 9.3%±0.2 and 10.2%±0.4 respectively. Cabbage moth 5mC content results the highest reported till now in insects and it is similar to the typical vertebrate one. Analysis of MspI and HpaII restriction pattern on M. brassicae DNA showed that a portion of its genome was methylated at CpG sites. Moreover, the absence of small digestion products after MspI digestion suggested that CpG are not clustered in the cabbage moth genome. Finally, methylation of repeated DNAs has been studied. Comparison of the restriction pattern of MspI and HpaII after hybridisation with the hobo, mariner, 28S and 5S rDNA probes did not evidence any difference indicating the absence of CpG methylation in all the studied repeated DNAs.     

Cytosine Methylation Changes in Rice Centromeric and Telomeric Sequences Induced by Foreign DNA Introgression

Cytosine methylation changes (hyper- or hypomethylation) in centromeric and telomeric sequences were observed in all three studied rice introgression lines containing DNA from wild rice, Zizania latifolia Griseb. The changed genomic Southern hybridization patterns were complex and non-concordant between a pair of isoschizomers (HpaII/MspI) digests, indicating methylation modifications at both the inner and outer cytosines of the CCGG sites. The changed patterns were inherited through generations. Possible mechanism for the methylation changes and their potential implications for the phenotypic variation and genome organization are discussed.     

Characterization of the level,target sites and inheritance of cytosine methylation in tomato nuclear DNA

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mCT. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.