Effects of single moor baths on physiological stress response and psychological state: a pilot study.

Moor mud applications in the form of packs and baths are widely used therapeutically as part of balneotherapy. They are commonly given as therapy for musculoskeletal disorders, with their thermo-physical effects being furthest studied. Moor baths are one of the key therapeutic elements in our recently developed and evaluated 3-week prevention program for subjects with high stress level and increased risk of developing a burnout syndrome. An embedded pilot study add-on to this core project was carried out to assess the relaxing effect of a single moor bath. During the prevention program, 78 participants received a total of seven moor applications, each consisting of a moor bath (42 °C, 20 min, given between 02:30 and 05:20 p.m.) followed by resting period (20 min). Before and after the first moor application in week 1, and the penultimate moor application in week 3, salivary cortisol was collected, blood pressure and heart rate were measured, and mood state (Multidimensional Mood State Questionnaire) was assessed. A Friedman test of differences among repeated measures was conducted. Post hoc analyses were performed using the Wilcoxon signed-rank test. A significant decrease in salivary cortisol concentration was seen between pre- and post-moor bath in week 1 (Z = -3.355, p = 0.0008). A non-significant decrease was seen between pre- and post-moor bath in week 3. Mood state improved significantly after both moor baths. This pilot study has provided initial evidence on the stress-relieving effects of single moor baths, which can be a sensible and recommendable therapeutic element of multimodal stress-reducing prevention programs. The full potential of moor baths still needs to be validated. A randomized controlled trial should be conducted comparing this balneo-therapeutic approach against other types of stress reduction interventions.

[Results of a Survey of Insurance Holders on Cure Regimes Performed in Foreign European Countries]. / Ergebnisse einer Versichertenbefragung zur Inanspruchnahme ambulanter Vorsorgeleistungen (Kurmaßnahmen) im europäischen Ausland.

OBJECTIVE: The aim of this study was to investigate the reasons of the insured in Germany for taking cure regimes in foreign European countries. METHODS: Health insurances conducted a survey including insurance holders in Germany who had taken a cure regime in a foreign European country in 2012 or 2011. In a paper-pencil interview, the sample was asked why they performed the cure regime abroad, how satisfied they were regarding the quality of the health professionals and the treatment, respectively, how long a positive effect lasted, where they spent their cure regime and about their plans regarding a future treatment in a health resort. RESULTS: In total, 443 insurance holders (60.7% female, mean age 68 years) were included. Price-performance ratio of board, lodging and treatment (75.6%), the health resorts' reputation (64.5%) and recommendations of the personal environment (56.1%) were the main reasons for taking a cure regime abroad. Most of the participants were satisfied with the quality of the health professionals and the health resort; two thirds rated the effect as lasting longer than 3 months. The study population predominantly took services of health resorts located in the Czech Republic, in Hungary and in Poland. For taking a cure in the future, most of the study participants (77%) preferred health resorts abroad, however, a gender-specific analysis revealed this trend to be more pronounced in the male study population. CONCLUSION: Based on the results of the survey one might suggest that cost factors along with high level of satisfaction regarding the quality of treatments provided at the health resorts abroad, positively influence the decision for taking a cure regime in a foreign country. In the sample of this study, the decision is rarely based on the health insurances' or the health resorts' promotion activity, respectively. However, to conclude on the total population, requires data of a representative sample.

New genomic resources for the honey bee(Apis mellifera L): development of a deep-coverage BAC library and a preliminary STC database

We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning enzyme HindIII in order to develop resources for structural genomics research. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and 2 empty vectors. Based on an estimated genome size of 270 Mb, this library provides approximately 15 haploid genome equivalents, allowing >99 probability of recovering any specific sequence of interest. High-density colony filters were gridded robotically using a Genetix Q-BOT in a 4 x 4 double-spotted array on 22.5-cm2 filters. Screening of the library with four mapped honey bee genomic clones and two bee cDNA probes identified an average of 21 positive signals per probe, with a range of 7-38 positive signals per probe. An additional screening was performed with nine aphid gene fragments and one Drosophila gene fragment resulting in seven of the nine aphid probes and the Drosophila probe producing positive signals with a range of 1 to 122 positive signals per probe (average of 45). To evaluate the utility of the library for sequence tagged connector analysis, 1152 BAC clones were end sequenced in both forward and reverse directions, giving a total of 2061 successful reads of high quality. End sequences were queried against SWISS-PROT, insect genomic sequence GSS, insect EST, and insect transposable element databases. Results in spreadsheet format from these searches are publicly available at the Clemson University Genomics Institute (CUGI) website in a searchable format (http://www.genome.clemson.edu/projects/stc/bee/AM__Ba/)

Construction of BAC libraries from two apomictic grasses to study the microcolinearity of their apospory-specific genomic regions.

We have constructed bacterial artificial chromosome (BAC) libraries from two grass species that reproduce by apospory, a form of gametophytic apomixis. The library of an apomictic polyhaploid genotype (line MS228-20, with a 2C genome size of approximately 4,500 Mbp) derived from a cross between the obligate apomict, Pennisetum squamulatum, and pearl millet ( P. glaucum) comprises 118,272 clones with an average insert size of 82 kb. The library of buffelgrass ( Cenchrus ciliaris, apomictic line B-12-9, with a 2C genome size of approximately 3,000 Mbp) contains 68,736 clones with an average insert size of 109 kb. Based on the genome sizes of these two lines and correcting for the number for false-positive and organellar clones, library coverages were found to be 3.7 and 4.8 haploid genome equivalents for MS 228-20 and B12-9, respectively. Both libraries were screened by hybridization with six SCARs (sequence-characterized amplified regions), whose tight linkage in a single apospory-specific genomic region had been previously demonstrated in both species. Analysis of these BAC clones indicated that some of the SCAR markers are actually amplifying duplicated regions linked in coupling in both genomes and that restriction enzyme mapping will be necessary to sort out the duplications.

New genomic resources for the honey bee(Apis mellifera L.): development of a deep-coverage BAC library and a preliminary STC database.

We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning enzyme HindIII in order to develop resources for structural genomics research. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and 2% empty vectors. Based on an estimated genome size of 270 Mb, this library provides approximately 15 haploid genome equivalents, allowing >99% probability of recovering any specific sequence of interest. High-density colony filters were gridded robotically using a Genetix Q-BOT in a 4 x 4 double-spotted array on 22.5-cm2 filters. Screening of the library with four mapped honey bee genomic clones and two bee cDNA probes identified an average of 21 positive signals per probe, with a range of 7-38 positive signals per probe. An additional screening was performed with nine aphid gene fragments and one Drosophila gene fragment resulting in seven of the nine aphid probes and the Drosophila probe producing positive signals with a range of 1 to 122 positive signals per probe (average of 45). To evaluate the utility of the library for sequence tagged connector analysis, 1152 BAC clones were end sequenced in both forward and reverse directions, giving a total of 2061 successful reads of high quality. End sequences were queried against SWISS-PROT, insect genomic sequence GSS, insect EST, and insect transposable element databases. Results in spreadsheet format from these searches are publicly available at the Clemson University Genomics Institute (CUGI) website in a searchable format (http://www.genome.clemson.edu/projects/stc/bee/AM__Ba/).

A framework for sequencing the rice genome.

Rice is an important food crop and a model plant for other cereal genomes. The Clemson University Genomics Institute framework project, begun two years ago in anticipation of the now ongoing international effort to sequence the rice genome, is nearing completion. Two bacterial artificial chromosome (BAC) libraries have been constructed from the Oryza sativa cultivar Nipponbare. Over 100,000 BAC end sequences have been generated from these libraries and, at a current total of 28 Mbp, represent 6.5% of the total rice genome sequence. This sequence information has allowed us to draw first conclusions about unique and redundant rice genomic sequences. In addition, more than 60,000 clones (19 genome equivalents) have been successfully fingerprinted and assembled into contigs using FPC software. Many of these contigs have been anchored to the rice chromosomes using a variety of techniques. Hybridization experiments have shown these contigs to be very robust. Contig assembly and hybridization experiments have revealed some surprising insights into the organization of the rice genome, which will have significant repercussions for the sequencing effort. Integration of BAC end sequence data with anchored contig information has provided unexpected revelations on sequence organization at the chromosomal level.

Melon bacterial artificial chromosome (BAC) library construction using improved methods and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2).

Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that co-segregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.

Physical mapping of the barley stem rust resistance gene rpg4.

The barley stem rust resistance gene rpg4 was physically and genetically localized on two overlapping BAC clones covering an estimated 300-kb region of the long arm of barley chromosome 7(5H). Initially, our target was mapped within a 6.0-cM region between the previously described flanking markers MWG740 and ABG391. This region was then saturated by integrating new markers from several existing barley and rice maps and by using BAC libraries of barley cv. Morex and rice cv. Nipponbare. Physical/genetic distances in the vicinity of rpg4 were found to be 1.0 Mb/cM, which is lower than the average for barley (4 Mb/cM) and lower than that determined by translocation breakpoint mapping (1.8 Mb/cM). Synteny at high resolution levels has been established between the region of barley chromosome 7(5H) containing the rpg4 locus and the subtelomeric region of rice chromosome 3 between markers S16474 and E10757. This 1.7-cM segment of the rice genome was covered by two overlapping BAC clones, about 250 kb of total length. In barley the markers S16474 and E10757 genetically delimit rpg4, lying 0.6 cM distal and 0.4 cM proximal to the locus, respectively.

Rice transposable elements: a survey of 73,000 sequence-tagged-connectors.

As part of an international effort to sequence the rice genome, the Clemson University Genomics Institute is developing a sequence-tagged-connector (STC) framework. This framework includes the generation of deep-coverage BAC libraries from O. sativa ssp. japonica c.v. Nipponbare and the sequencing of both ends of the genomic DNA insert of the BAC clones. Here, we report a survey of the transposable elements (TE) in >73,000 STCs. A total of 6848 STCs were found homologous to regions of known TE sequences (E<10(-5)) by FASTX search of STCs against a set of 1358 TE protein sequences obtained from GenBank. Of these TE-containing STCs (TE-STCs), 88% (6027) are related to retroelements and the remaining are transposase homologs. Nearly all DNA transposons known previously in plants were present in the STCs, including maize Ac/Ds, En/Spm, Mutator, and mariner-like elements. In addition, 2746 STCs were found to contain regions homologous to known miniature inverted-repeat transposable elements (MITEs). The distribution of these MITEs in regions near genes was confirmed by EST comparisons to MITE-containing STCs, and our results showed that the association of MITEs with known EST transcripts varies by MITE type. Unlike the biased distribution of retroelements in maize, we found no evidence for the presence of gene islands when we correlated TE-STCs with a physical map of the CUGI BAC library. These analyses of TEs in nearly 50 Mb of rice genomic DNA provide an interesting and informative preview of the rice genome.

[Perceptions and practices related with tuberculosis and treatment compliance in Chiapas, Mexico]. / Percepciones y prácticas relacionadas con la tuberculosis y la adherencia al tratamiento en Chiapas, México.

OBJECTIVE: To identify health perceptions and practices and non-adherence to therapy among tuberculosis patients. MATERIAL AND METHODS: Qualitative research work consisting of 11 group interviews with 62 tuberculosis patients during 1997-1998 in the Central, Highlands, and Border Regions of Chiapas, Mexico. RESULTS: Perceived causes of tuberculosis included contagion via food utensils, excess work, malnutrition, and cold, as well as other causes unrelated to person-to-person contagion. The resulting incapability to work resulted in an economic crisis for both the patients and their family members. As a result of the social stigma imposed by the disease, patients perceived a negative impact on their personal life, family, work, and community. CONCLUSIONS: Lack of knowledge regarding tuberculosis is an important factor in the selection of and adherence to different care alternatives. Inadequate care provided by health services, including an unsatisfactory physician-patient relationship, resulted in diagnostic delay and non-adherence to therapy. Education programs to promote basic knowledge regarding tuberculosis and its treatment are necessary in this region.

A bacterial artificial chromosome library for sugarcane.

Modern cultivated sugarcane is a complex aneuploid polyploid with an estimated genome size of 3000 Mb. Although most traits in sugarcane show complex inheritance, a rust locus showing monogenic inheritance has been documented. In order to facilitate cloning of the rust locus, we have constructed a bacterial artificial chromosome (BAC) library for the cultivar R570. The library contains 103,296 clones providing 4.5 sugarcane genome equivalents. A random sampling of 240 clones indicated an average insert size of 130 kb allowing a 98% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4 × 4 double-spotted array on 22.5-cm(2) filters. Each set of five filters provides a genome coverage of 4x with 18,432 clones represented per filter. Screening of the library with three different barley chloroplast gene probes indicated an exceptionally low chloroplast DNA content of less than 1%. To demonstrate the library's potential for map-based cloning, single-copy RFLP sugarcane mapping probes anchored to nine different linkage groups and three different gene probes were used to screen the library. The number of positive hybridization signals resulting from each probe ranged from 8 to 60. After determining addresses of the signals, clones were evaluated for insert size and HindIII-fingerprinted. The fingerprints were then used to determine clone relationships and assemble contigs. For comparison with other monocot genomes, sugarcane RFLP probes were also used to screen a Sorghum bicolor BAC library and two rice BAC libraries. The rice and sorghum BAC clones were characterized for insert size and fingerprinted, and the results compared to sugarcane. The library was screened with a rust resistance RFLP marker and candidate BAC clones were subjected to RFLP fragment matching to identify those corresponding to the same genomic region as the rust gene.

[Obstacles to overcome in the control of pulmonary tuberculosis in the border region of Chiapas, Mexico]. / Retos a superar en el control de la tuberculosis pulmonar en la región fronteriza de Chiapas, México.

OBJECTIVE: To improve the control of the pulmonary tuberculosis in the Border Region of Chiapas, Mexico. DESIGN: Academic researchers, health development workers from the nongovernmental sector and government health authorities met in a workshop to analyze recent experiences with tuberculosis. RESULTS: Among the important issues addressed were: with regard to official health services, the lack of resources, particularly medication, organizational problems which result in poor or absent communication within and among different health entities, the under diagnosis of cases and the lack of sufficient index of suspicion for tuberculosis among health personnel. With regard to the population at risk, there are profound socio-cultural barriers which include a lack of confidence in the quality of government health care centers and little attention given to chronic cough. Poorest, indigenous and more remote people have less access to care and are more likely to have advanced tuberculosis before seeking treatment if at all. New strategies proposed were to integrate communication efforts in tuberculosis control among all the involved health services, including private physicians, identify those patients at greatest risk, improve diagnostic skills of health providers, develop education campaigns in rural areas. CONCLUSIONS: Certain factors which impede better TB control seem amenable to change, others, such as severe poverty, particularly among peasants and indigeneous people, as well as the current political disruption, will require much broader intersectorial interventions.

Single-amino acid substitutions eliminate lysine inhibition of maize dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) catalyzes the first step in biosynthesis of lysine in plants and bacteria. DHPS in plants is highly sensitive to end-product inhibition by lysine and, therefore, has an important role in regulating metabolite flux into lysine. To better understand the feedback inhibition properties of the plant enzyme, we transformed a maize cDNA for lysine-sensitive DHPS into an Escherichia coli strain lacking DHPS activity. Cells were mutagenized with ethylmethanesulfonate, and potential DHPS mutants were selected by growth on minimal medium containing the inhibitory lysine analogue S-2-aminoethyl-L-cysteine. DHPS assays identified surviving colonies expressing lysine-insensitive DHPS activity. Ten single-base-pair mutations were identified in the maize DHPS cDNA sequence; these mutations were specific to one of three amino acid residues (amino acids 157, 162, and 166) localized within a short region of the polypeptide. No other mutations were present in the remaining DHPS cDNA sequence, indicating that altering only one of the three residues suffices to eliminate lysine inhibition of maize DHPS. Identification of these specific mutations that change the highly sensitive maize DHPS to a lysine-insensitive isoform will help resolve the lysine-binding mechanism and the resultant conformational changes involved in inhibition of DHPS activity. The plant-derived mutant DHPS genes may also be used to improve nutritional quality of maize or other cereal grains that have inadequate lysine content when fed to animals such as poultry, swine, or humans.

Efecto de las prácticas anticonceptivas sobre la fecundidad en la región fronteriza de Chiapas, México / The effect of contraception on fertillity in the border region of Chiapas, México

Objetivo. Estimar el efecto de las prácticas anticonceptivas sobre la fecundidad en la región fronteriza de Chiapas, México. Material y métodos. Durante 1994 se realizó un estudio epidemiológico de corte transversal en una muestra representativa de 1 560 mujeres no indígenas de 15 a 49 años en la región fronteriza de Chiapas. Se estimaron la prevalencia de uso de métodos anticinceptivos y las tasas globales de fecundidad (TGF) por tamaño de la localidad de residencia (rurales, intermedias y urbanas). Se comparó la TGF entre las mujeres nunca usuarias y alguna vez usuarias de métodos anticonceptivos. Resultados. La TGF estimada para la región fue de 3.67 y varió de 4.14 para áreas rurales a 3.36 para áreas urbanas. No se observaron diferencias en la TGF (3.74 y 3.88) ni el promedio de hijos nacidos vivos (3.47 y 3.48) entre usuarios alguna vez y nunca usuarias de métodos anticonceptivos. Conclusiones. El mayor efecto de las prácticas anticonceptivas en la fecundidad se observó en áreas rurales. El uso tardió de métodos anticonceptivos, así como la edad de unión más temprana entre las usuarias son factores que influyen para dar por resultado un escaso efecto en la fecundidad

Objetive.To estimate the effect of contraception on fertility in the border region of Chiapas, Mexico. Materials and methods. In 1994 an epidemiological cross-sectional study was carried out on a representative sample of 1 560 non-indigenous women between ages 15 and 49 in the border region of Chiapas. The prevalence of contraception practices and the total fertility rates (TER) were obtained and stratified by rural, intermediate and urban communities. TER were compared between women who had never used contraceptives and those who had used them. Results. The estimated TFR was 3.67 and varied from 4.14 in rural areas to 3.36 in urban areas. There were no differences in the TFR (3.74 and 3.88) nor in the average live births (3.47 and 3.48) between women who had never used contraceptives and those who had used them. Conclusions. The major effect of contraception on fertility was observed in rural areas. Factors which influence the small impact of contraception on fertility include the late use of these methods and the early age of first union among users.

[The effect of contraception on fertility in the border region of Chiapas, Mexico]. / Effecto de las prácticas anticonceptivas sobre la fecundidad en la región fronteriza de Chiapas, México.

OBJECTIVE: To estimate the effect of contraception on fertility in the border region of Chiapas, Mexico. MATERIALS AND METHODS: In 1994 an epidemiological cross-sectional study was carried out on a representative sample of 1,560 non-indigenous women between ages 15 and 49 in the border region of Chiapas. The prevalence of contraception practices and the total fertility rates (TFR) were obtained and stratified by rural, intermediate and urban communities. TFR were compared between women who had never used contraceptives and those who had used them. RESULTS: The estimated TFR was 3.67 and varied from 4.14 in rural areas to 3.36 in urban areas. There were no differences in the TFR (3.74 and 3.88) nor in the average live births (3.47 and 3.48) between women who had never used contraceptives and those who had used them. CONCLUSIONS: The major effect of contraception on fertility was observed in rural areas. Factors which influence the small impact of contraception on fertility include the late use of these methods and the early age of first union among users.

PIP: The objective of this study was to estimate the effect of contraception on fertility in the border region of Chiapas, Mexico. In 1994, an epidemiological cross-sectional study was carried out on a representative sample of 1560 nonindigenous women between the ages of 15 and 49 years in the border region of Chiapas. The prevalence of contraception practices and the total fertility rates (TFR) were obtained and stratified by rural, intermediate, and urban communities. TFR were compared between women who had never used contraceptives and those who had used them. The estimated TFR was 3.67 and varied from 4.14 in rural areas to 3.36 in urban areas. There were no differences in the TFR (3.74 and 3.88) nor in the average live births (3.47 and 3.48) between women who had never used contraceptives and those who had used them. The major effect of contraception on fertility was observed in rural areas. Factors which influence the small impact of contraception on fertility include the late use of these methods and the early age of first union among users. (author's)

Cell Ablation Reveals That Expression from the Phaseolin Promoter Is Confined to Embryogenesis and Microsporogenesis.

Most previous studies of the [beta]-phaseolin (phas) gene, which encodes the major storage protein in bean (Phaseolus vulgaris L.), have shown its expression to be rigorously confined to the developing seed, both in bean and transgenic tobacco (Nicotiana tabacum L. cv Xanthi) plants. To confirm unequivocally the lack of phas expression in vegetative tissues, we placed the diphtheria toxin A-chain (DT-A) coding region under the control of [beta]-phaseolin promoter sequences. Tobacco plants transgenic for phas/DT-A were phenotypically normal until flowering, when they produced anthers that were externally normal but contained no viable pollen. Microscopic examination of immature anthers revealed a normal tapetum, but the pollen mother cells did not undergo meiosis and subsequently degenerated, resulting in male-sterile plants. This demonstration of phas expression during microsporogenesis was corroborated by the expression of [beta]-glucuronidase in pollen of plants transformed with comparable phas/uidA constructs. Although these findings suggested that similarities in phas expression may exist between seed and pollen maturation, no phas activity could be detected in bean pollen. After fertilization of the DT-A-transformed plants with pollen from wild-type tobacco, 50% of the resulting embryos aborted at the heart stage, defining this as the earliest time for phas expression during embryogenesis.

[Risk factors for antitubercular drug resistance in Chiapas, Mexico]. / Factores de riesgo para resistencia a drogas antifímicas en Chiapas, México.

OBJECTIVES: To determine risk factors for antibiotic resistance in patients with pulmonary tuberculosis in four Health Jurisdictions of the state of Chiapas. MATERIAL AND METHODS: A case-control study was carried out in patients diagnosed by acid fast smear during 1992. A questionnaire was applied which included variables related to the diagnosis, treatment and follow-up of the patients. Sputum samples were collected for culture and sensitivity tests. A case of drug-resistant pulmonary tuberculosis was defined as the presence of culture colonies showing resistance to one or more drugs. The control group was patients with negative smears and cultures or positive cultures for M. tuberculosis sensitive to the specific drugs. RESULTS: Of the total of 18 individuals reported to have positive cultures, 13 (72.2%) were resistant to one or more drugs. Resistance to two or more drugs was found in 10 of them of which three were resistant to five antituberculosis drugs. The most frequent resistance was to isoniazid (77%). Risk factors for resistance encountered in this patient population were monotherapy (OR = 34.2), abandonment of treatment (OR = 6.86), a prolonged period of illness (OR = 6.40), delay in diagnosis and a history of prior therapy (OR = 28.3). CONCLUSIONS: The high proportion of patients resistant to antituberculosis therapy poses a public health problem and is a clear consequence of the problems arising from inadequate treatment.

Complete sequence of the binary vector Bin 19.

Despite the widespread use of Bin 19 as a vector for plant transformation, detailed sequence information on its T-DNA region has only recently become available. We now show that the non-T-DNA region, like the T-DNA region, contains several superfluous insertions and find that some functional elements may not contain optimal sequences. Knowledge of the complete 11,777 bp sequence will aid in the construction of exceptionally efficient derivative vectors of approximately half this size. Precise knowledge of restriction sites and removal of unnecessary sequences will facilitate plasmid manipulations and plant transformation.