Single Enzyme Digestion Research Articles

Virtual digest identification of secondary enzymes for use in pulsed-field gel electrophoresis of Staphylococcus aureus

Pulsed-field gel electrophoresis (PFGE) is currently the gold standard for methicillin-resistant Staphylococcus aureus (MRSA) typing but only one enzyme, SmaI, is currently used for restriction digest. We report the use of virtual digestion to identify enzymes for S. aureus PFGE. Two enzymes ( EagI and SacII) were identified and successfully used to characterize two sets of S. aureus isolates, 12 USA300, and 14 additional MRSA isolates comprised of seven SmaI patterns. Phylogenetic analysis of patterns generated by all enzymes determined that the USA300 MRSAs are identical. In contrast, digestion with EagI or SacII resolved one to two band differences among three MRSA pattern sets that were not detected using SmaI. These results demonstrate that a second enzyme may detect differences in S. aureus isolates not detected by single enzyme digestion. However, because isolates differing by one to two bands are considered identical, such discrimination may not be clinically or epidemiologically relevant.

First Report of 16S rDNA II Group Phytoplasma on Polygala mascatense, a Weed in Oman

Polygala mascatense Boiss. (family Polygalaceae) is a common weed found in neglected farms, under date palm trees, and in stony locations throughout the Sultanate of Oman (1). It is a perennial herb approximately 30 to 40 cm tall, has slender branches, is woody at the base, and has linear leaves with purple flowers. Recently (November 2004), in the interior region of Oman (210 km south of Muscat), some polygala plants were found stunted with small leaves, bushy growth, and the floral parts were showing phyllody symptoms. Total genomic DNA extracted from asymptomatic and symptomatic plants with modified cetyltrimethylammoniumbromide (CTAB) buffer method (4) was used as a template for direct polymerase chain reaction (PCR) amplification of phytoplasma 16S rDNA with P1/P7 primers. Direct PCR product was used as template DNA for nested PCR with primers R16F2n/R16R2. DNA from plants infected with alfalfa and lime witches'-broom phytoplasma was used as positive controls, and DNA from healthy plants and water was used as negative controls. Products from nested PCR (1.2 kb) were analyzed by using single endonuclease enzyme digestion (restriction fragment length polymorphism [RFLP]) with Tru9I, HaeIII, HhaI, TaqI, AluI, and RsaI (3). The results showed the presence of a 1.8-kb product amplified with direct PCR and a 1.2-kb product of the nested PCR from infected polygala and the positive controls, whereas no PCR products were observed in the negative controls. The PCR assay confirmed the presence of phytoplasma causing witches'-broom disease in polygala. The RFLP results showed the polygala phyto-plasma to be most similar to the alfalfa phytoplasma, a member of 16SrII group (2). Infected polygala weeds may serve as a reservoir for alfalfa witches'-broom phytoplasma that causes annual losses over $25 million to alfalfa cultivation in Oman (2). A detailed investigation needs to be carried out to establish transmission of phytoplasma from polygala to alfalfa. To our knowledge, this is the first report of phytoplasma infecting polygala weeds in Oman.

Identification of Africanized Honey Bee (Hymenoptera: Apidae) Mitochondrial DNA: Validation of a Rapid Polymerase Chain Reaction-Based Assay

Polymerase chain reaction (PCR)-amplified mitochondrial DNA (mtDNA) assays have been used in studies of the Africanization process in neotropical feral and managed honey bee populations. The approach has been adopted, in conjunction with morphometric analysis, to identify Africanized bees for regulatory purposes in the United States such as in California. In this study, 211 Old World colonies, representing all known introduced subspecies in the United States, and 451 colonies from non-Africanized areas of the southern United States were screened to validate a rapid PCR-based assay for identification of Africanized honey bee mtDNA. This PCR-based assay requires a single enzyme digestion (BglII) of a single PCR-amplified segment of the cytochrome b gene. The BglII polymorphism discriminates the mitochondrial haplotype (mitotype) of Apis mellifera scutellata L. (ancestor of Africanized bees) from that of A. m. mellifera, A. m. caucasia, A. m. ligustica, A. m. carnica, A. m. lamarcki, A. m. cypria, A. m. syriaca, and some A. m. iberiensis, but not from that of A. m. intermissa and some A. m. iberiensis. Nonetheless, given the very low frequency (<1%) of African non-A. m. scutellata mitotype present before arrival of Africanized bees in the United States, cytochrome b/BglII assay can be used to identify maternally Africanized bees with a high degree of reliability.

O-Glycan Sialylation and the Structure of the Stalk-like Region of the T Cell Co-receptor CD8

Studies of mucins suggest that the structural effects of O-glycans are restricted to steric interactions between peptide-linked GalNAc residues and adjacent polypeptide residues. It has been proposed, however, that differential O-glycan sialylation alters the structure of the stalk-like region of the T cell co-receptor, CD8, and that this, in turn, modulates ligand binding (Daniels, M. A., Devine, L., Miller, J. D., Moser, J. M., Lukacher, A. E., Altman, J. D., Kavathas, P., Hogquist, K. A., and Jameson, S. C. (2001) Immunity 15, 1051-1061; Moody, A. M., Chui, D., Reche, P. A., Priatel, J. J., Marth, J. D., and Reinherz, E. L. (2001) Cell 107, 501-512). We characterize the glycosylation of soluble, chimeric forms of the alphaalpha- and alphabeta-isoforms of murine CD8 containing the O-glycosylated stalk of rat CD8alphaalpha, and we show that the stalk O-glycans are differentially sialylated in CHO K1 versus Lec3.2.8.1 cells (82 versus approximately 6%, respectively). Sedimentation analysis indicates that the Perrin functions, Pexp, which reflect overall molecular shape, are very similar (1.61 versus 1.54), whereas the sedimentation coefficients (s) of the CHO K1- and Lec3.2.8.1-derived proteins differ considerably (3.73 versus 3.13 S). The hydrodynamic properties of molecular models also strongly imply that the sialylated and non-sialylated forms of the chimera have parallel, equally highly extended stalks ( approximately 2.6 A/residue). Our analysis indicates that, as in the case of mucins, the overall structure of O-glycosylated stalk-like peptides is sialylation-independent and that the functional effects of differential CD8 O-glycan sialylation need careful interpretation.

First Report of Alder Yellows Phytoplasma in the Eastern Baltic Region.

Alnus glutinosa (alder) is widespread in Europe and is an important component of biological diversity in natural forest ecosystems in the Baltic Region. In 2000, diseased trees of A. glutinosa exhibiting characteristically phytoplasmal disease symptoms of shoot proliferation and leaf yellowing were observed in Aukstaitija National Park, Lithuania. In other parts of Europe, alder is affected by a phytoplasmal disease known as alder yellows, which is characterized by symptoms that include yellowing and reduced leaf size, die-back of branches, and decline of trees (2,3). Proliferation of shoots has not been previously reported with this disease. An association between alder yellows and infection by a phytoplasma has been reported in A. glutinosa in Germany and Italy, and a phytoplasma has been found in A. glutinosa in France and Hungary (2,4). We examined symptomatic alder from Lithuania using nested polymerase chain reaction (PCR) (1), primed by P1/P7 and followed by R16F2n/R16R2 (F2n/R2), for amplification of phytoplasmal ribosomal (r) DNA. The results indicated the presence of a phytoplasma, designated ALY-L, in the diseased alder. We classified the ALY-L phytoplasma through restriction fragment length polymorphism (RFLP) analysis of 16S rDNA. A 1.2-kbp fragment (F2n-R2 segment) of rDNA, amplified in PCR primed by F2n/R2, was analyzed using single endonuclease enzyme digestion with AluI, MseI, KpnI, HhaI, HaeIII, HpaI, HpaII, RsaI, HinfI, TaqI, Sau3AI, BfaI, and ThaI. On the basis of collective RFLP patterns, phytoplasma ALY-L was classified as a member of group 16SrV (group V, elm yellows group), subgroup C. The amplified 16S rDNA was cloned in Escherichia coli and sequenced, and the sequence was deposited in the GenBank data library (Accession No. AY028789). Nucleotide sequence alignment revealed that 16S rDNA from phytoplasma ALY-L shared 100% sequence similarity with 16S rDNA (GenBank Accession No. Y16387) from a phytoplasma associated with alder yellows (ALY) disease in Italy. The results support the conclusion that a strain of ALY phytoplasma is present in Lithuania. Phytoplasmas belonging to groups 16SrI (aster yellows phytoplasma group) and III (X-disease phytoplasma group) have been found in herbaceous plant species in Lithuania. This report records the first finding of a group V phytoplasma, and the first finding of a phytoplasma in a tree species in the eastern Baltic Region. These findings contribute knowledge about the diversity of phytoplasmas in the Baltic Region and the distribution of ALY phytoplasma in Europe. Apparently, A. glutinosa may be infected by the phytoplasma but not develop obvious disease symptoms, as has been reported elsewhere (3). Thus, it is possible that ALY-L phytoplasma is widespread, but as yet undetected, throughout the geographic range of alder in the Baltic Region. This possibility is supported by the finding of the monophagous leafhopper vector (Oncopsis alni) of ALY phytoplasma throughout Europe (cited in Maixner and Reinert [3]). Further research is needed to assess the impact of phytoplasmal infections such as those by ALY-related phytoplasma strains on trends in biological diversity in the natural forest ecosystems of the Baltic Region and elsewhere in Europe.

First Report of Aster Yellows-Related Subgroup I-A Phytoplasma Strains in Carrot, Phlox, Sea-Lavender, Aconitum, and Hyacinth in Lithuania.

Phytoplasma strains that belong to group 16SrI (aster yellows phytoplasma group), subgroup A (I-A, North American tomato big bud phytoplasma subgroup) were discovered in diverse plant species in Lithuania. Plants in which the strains were found exhibited symptoms characteristic of infections by phytoplasma. Carrot (Daucus sativus) with carrot proliferation disease exhibited symptoms of proliferation of the crown, chlorosis of young leaves, and reddening of mature leaves. Diseased phlox (Phlox paniculata) exhibited symptoms of virescence and leaf chlorosis. Diseased sea-lavender (Limonium sinuatum) exhibited abnormal proliferation of shoots, chlorosis of young leaves, reddening of mature leaves, and degeneration of flowers. Diseased hyacinth (Hyacinthus orientalis) exhibited chlorosis of leaves and undeveloped flowers. Diseased Aconitum sp. exhibited proliferation of shoots. Phytoplasma-characteristic ribosomal (r) DNA was detected in the plants by use of the polymerase chain reaction (PCR). The rDNA was amplified in PCR primed by primer pair P1/P7 and reamplified in nested PCR primed by primer pair R16F2n/R16R2 (F2n/R2), as previously described (1). The phytoplasmas were classified through restriction fragment length polymorphism (RFLP) analysis of 16S rDNA, amplified in the nested PCR primed by F2n/R2, using single endonuclease enzyme digestion with AluI, MseI, KpnI, HhaI, HaeIII, HpaI, HpaII, RsaI, HinfI, TaqI, and Sau3AI. Collective RFLP patterns indicated that all detected phytoplasma strains were affiliated with subgroup I-A. The 16S rDNA amplified from the phytoplasma (CarrP phytoplasma) in diseased carrot was cloned in Escherichia coli, sequenced, and the sequence deposited in the GenBank data library (GenBank accession no. AF291682). The 16S rDNAs of CarrP and tomato big bud (GenBank acc. no. AF222064) phytoplasmas shared 99.8% nucleotide sequence similarity. Phytoplasmas belonging to group 16SrIII (3), group 16SrV (D. Valiunas, unpublished data), and subgroup I-C in group 16SrI (2,3) occur in Lithuania. This report records the first finding of a subgroup I-A phytoplasma in the Baltic region and expands the known plant host range of this phytoplasma subgroup.

Sporadic pheochromocytomas are rarely associated with germline mutations in the vhl tumor suppressor gene or the ret protooncogene.

Pheochromocytoma is a tumor that may occur sporadically or may be a manifestation of a hereditary disease, such as von Hippel-Lindau disease (VHL) and multiple endocrine neoplasia (MEN) type 2. As patients with VHL or MEN type 2 are at risk to develop multiple tumors, they must be distinguished from sporadic cases. We determined the incidence of VHL and MEN type 2 among 62 German patients diagnosed with pheochromocytoma without a history of a hereditary disease. Germline analyses of the vhl gene and exons 10, 11, and 13 of the ret protooncogene were performed by PCR, single strand conformation polymorphism, enzyme digestion, or sequencing. Two patients (3%) showed vhl mutations (95% confidence interval, 1-11%). One patient showed loss of the MspI restriction site at nucleotides 712/713. Another patient had a C/T change at an intronic site that was also detected in 2 of his offspring. No mutations were detected in the ret protooncogene (97.5% confidence interval, 0-6%). In Germany, most sporadic pheochromocytomas are not due to VHL or MEN type 2. Therefore, clinical work-up in patients with pheochromocytoma without signs of hereditary disease is not recommended. However, because the costs of genetic screening are relatively low, and each index case allows optimal care for family members, molecular testing might be cost-effective.

The 5'-untranslated region sequence of a potential new genotype of bovine viral diarrhea virus.

The 5' untranslated region (UTR) of several bovine viral diarrhea virus (BVDV) isolates from the severe Quebec outbreak was amplified by polymerase chain reaction (PCR) and sequenced. Sequences revealed the loss, for the BVDV type II isolates, of an internal PstI restriction site, which is present in all known BVDV type 1 5' UTR sequences. A single restriction enzyme digestion (PstI) of an aliquot of PCR product allowed us to differentiate BVDV type I and BVDV type II.

Identification of a set of RFLP probes for subspecies differentiation in Oryza sativa L.

Sixty-eight indica-japonica tester-differentiating RFLP probes were tested in seven indica and seven japonica varieties of rice (Oryza sativa L.) with four enzyme digestions (EcoRI, EcoRV, HindIII and DraI). Twenty-one DNA clones were isolated as indica-japonica subspecies-differentiating probes. A set of 13 probes was established as core probes for subspecies differentiation and a pooled blotting analysis was carried out to facilitate the application of RFLP in rice genetics and breeding practice. A dendrogram of 12 wide-compatibility varieties was constructed based on RFLPs detected by 13 core probes with single enzyme digestions. It was speculated that most RFLPs of indica-japonica differentiating probes were generated by insertions/deletions, which may be of great significance for the origin and differentiation of subspecies in Oryza sativa L.

Organisation of the plastid genome from the rhodophyteChondrus crispus(Gigartinales); sequence and phylogeny of the 16S rRNA gene

The plastid genome of the rhodophyte Chondrus crispus was characterised by restriction mapping and hybridisation analysis. As deduced from single and double enzyme digests, the plastid genome is circular with a molecular size of approximately 173 kilobases. The genome appears to have only one copy of the 16S rRNA gene. The nucleotide sequence of this gene and flanking regions was determined. Its length was estimated to be 1491 nucleotides. Its secondary structure was constructed and compared with other known 16S rRNA secondary structures from cyanobacteria and plastids. A phylogenetic tree was built upon alignment of various 16S rDNA sequences from eubacteria and from green, red and brown plastids. The results support the idea that heterokont and cryptophyte plastids have their origin in the ‘red algal’ radiation.

Genetics of streptomycin resistance in methicillin-sensitive multiply-resistant Staphylococcus aureus.

Streptomycin resistance was detected in 17 of 20 multiply-resistant Staphylococcus aureus isolates from a hospital in a southeastern Nigerian town. The isolates were uniformly sensitive to methicillin, erythromycin, gentamicin, mupirocin, ciprofloxacin and vancomycin but produced beta-lactamase and were resistant to other antimicrobial agents and harbored different plasmids which ranged in size and number from 1.0 to c, 40 kb and one to six respectively. Curing and transfer experiments demonstrated that streptomycin resistance (Smr) was located on plasmids in 15 of the 17 isolates. 16 Smr plasmids were isolated and characterized. They belonged to three distinct groups based on size and resistance phenotypes. Eight 4.4 kb plasmids encoded Smr only, three 4.7 kb plasmids encoded resistance to streptomycin and chloramphenicol (SmCm) and five 23.8 kb plasmids encoded resistance to streptomycin, kanamycin, neomycin, cadmium and beta-lactamase production (CPKNS). Restriction endonuclease analysis demonstrated that the 4.4 kb Smr plasmids were similar to one another and indistinguishable from pUB109, an incompatibility group 5 Smr plasmid, suggesting that they may also belong to incompatibility group 5. The SmCm and the CPKNS plasmid groups also gave identical restriction patterns with single and double enzyme digests. Further transfer experiments with one of the SmCm plasmids led to the isolation of a 4.4 kb Smr plasmid which was indistinguishable from the other 4.4 kb plasmids, suggesting that the SmCm plasmids are natural recombinants between a streptomycin and chloramphenicol resistance plasmid. The results demonstrate a multiple origin of streptomycin resistance in the S. aureus population studied.

Chloroplast DNA differences between cultivated hop, Humulus lupulus and the related species H. japonicus

Chloroplast DNA (cpDNA) of Humulus Lupulus and H. japonicus was examined by restriction endonuclease analysis with BamHI, BanI, BclI, BstEII, DraI, EcoRI, EcoRV, HindIII, KpnI, PaeR7I, PstI, PvuII, SalI and XhoI. The restriction fragment patterns showed that the cpDNAs shared a large number of restriction sites. However, the chloroplast genomes of the two species could be distinguished by differences in restriction site and restriction fragment patterns in the PstI, PvuII, BclI, EcoRV, DraI and HindIII digests. On the basis of the complexity of restriction enzyme patterns, the enzymes PstI, PvuII, SalI, KpnI and XhoI were selected for mapping the chloroplast genomes. Single and double restriction enzyme digests of cpDNA from the two species were hybridized to cpDNA probes of barley and tobacco. The data obtained from molecular hybridization experiments were used to construct the cleavage site maps. Except for the PstI digest, the arrangement of cpDNA restriction sites was found to be the same for both species. An extra PstI site was present in H. lupulus. Three small insertions/deletions of about 0.8 kbp each were detected in the chloroplast genomes of the two species. Two of these insertions/deletions were present in the large and one in the small singlecopy region of the chloroplast genome. The cpDNA of Humulus was found to be a circular molecule of approximately 148 kbp that contains two inverted repeat regions of 23 kbp each, a small and a large single -copy region of approximately 20 kbp and 81 kbp, respectively. The chloroplast genome of hop has the same physical and structural organization as that found in most angiosperms.

Chloroplast genome organization of bromegrass, Bromus inermis Leyss

A physical map of the Bromus inermis chloroplast genome was constructed using heterologous probes of barley and wheat chloroplast DNA (cpDNA) to locate restriction sites. The map was aligned from data obtained from filter hybridization experiments on single and double enzyme digests. Cleavage sites for the enzymes PstI, SalI, KpnI, XhoI and PvuII were mapped. The chloroplast genome of B. inermis is similar in physical organization to that of other grasses. The circular cpDNA molecule of B. inermis has the typical small (12.8 kbp) and large (81.3 kbp) single-copy regions separated by a pair of inverted repeat (21 kbp) regions. The cpDNA molecule of B. inermis is collinear in sequence to that of wheat, rye, barley and oats. No structural rearrangements or major deletions were observed, indicating that the cpDNA of Bromus is a useful tool in phylogenetic studies.

Differentiation of two rabies strains in Estonia with reference to recent Finnish isolates.

We compared 24 rabies samples collected in Estonia in 1989 to 1992, to identify the kinds of rabies strains circulating in this country. Eleven of the strains came from the islands of Saaremaa, Hiiumaa and Muhu, off the Baltic coast; 13 came from the mainland. The mainland strains, like those from the 1988 to 1989 epizootic in Finland, were antigenically different from the 11 island isolates. The island isolates reacted negatively with monoclonal antibody W-187.5 as does the SAD B19 rabies vaccine strain, currently spread as baits to wildlife in Finland and other parts of Europe. In order to unambiguously distinguish the island isolates from the SAD B19 vaccine, we developed a polymerase chain reaction (PCR) protocol for rabies, followed by a single restriction enzyme digestion. This method enabled the island isolates to be differentiated with ease from the vaccine strain SAD B19 at the level of the nucleoprotein-coding region. Additionally, this method had the ability to distinguish other polar field isolates examined, as well as the laboratory challenge virus strain CVS, from SAD B19 vaccine. Modifications of the above PCR method may be used for epidemiological investigations of new outbreaks or of outbreaks involving different species.

Use of cellular oncogene probes to identify Morone hybrids.

We tested the ability of cellular oncogene (c-onc) probes to identify F1 hybrids and the lineage of known backcrosses within the fish genus Morone. Total DNA was isolated from five to 14 individuals per North American Morone species (striped bass, white bass, white perch, and yellow bass). The DNA was digested with two restriction enzymes, Eco RI and Hin dIII, Southern blotted, and hybridized to six different c-onc probes including v-abl, v-erb B, c-myc, c-H-ras, c-K-ras, and v-src. We found fixed genotypic differences among the four species for all six probes in single restriction enzyme digests. The heritability of these nuclear DNA genotypes was evaluated in hatchery-produced F1 Morone hybrids (striped bass x white bass and striped bass x white perch) tested with the six informative single probe/restriction enzyme combinations. All F1 individuals exhibited heterozygosity in all diagnostic nuclear DNA fragments, confirming the Mendelian inheritance of these genotypes in these fish. Furthermore, analysis of these nuclear DNA genotypes in hatchery-produced backcrosses of F1 hybrids striped bass x (white bass x striped bass) detected both recombinant and parental genotypes at all six polymorphic c-onc sequences. The lineage of suspected Morone hybrids of unknown descent collected from Lewis Smith Lake, Alabama, and from the Occoquan River, Virginia, was determined using the c-onc probes. Our results suggest that c-onc probes are suitable markers to unequivocally identify F1 hybrids and backcrosses and to quantify introgression in natural populations of fishes. The addition of RFLP analysis of mtDNA provided a complete ancestral history of individual fish.

Discrimination among Atlantic Coast Populations of American Shad (Alosa sapidissima) Using Mitochondrial DNA

We used restriction endonuclease analysis of mitochondrial DNA (mtDNA) to differentiate among spawning stocks of American shad (Alosa sapidissima). Highly purified mtDNA was isolated from shad from four major spawning rivers: the St. John's (Florida), the Delaware, and the Hudson in the United States and the Miramichi in New Brunswick, Canada. Primarily four-and-five-base-cutting restriction enzymes were used to prepare both individual enzyme profiles and composite genotypes. Three separate spawning stocks, St. John's, Delaware–Hudson, and Miramichi, could be distinguished based on frequency differences in mtDNA genotypes generated by single restriction enzyme digests. We could not distinguish Delaware from Hudson River shad. Only a single definitive restriction site polymorphism was observed among all samples, but polyacrylamide gel electrophoretic mobility variants were common. Eco RI, Dde I, and Rsa I revealed stock-specific mtDNA genotypes. The frequencies of some genotypes occurred in latitudinal clines. Fifty-seven of 81 fish showed individual-specific composite genotypes. Geographic partitioning of genotypes suggests that mtDNA analysis may be useful for the identification of some American shad stocks and their relative contributions to mixed coastal fisheries.

Cell‐surface heparan sulfate and heparan‐sulfate/chondroitin‐sulfate hybrid proteoglycans of mouse mammary epithelial cells

The hydrophobic cell-surface proteoglycans of mouse mammary epithelial cells were purified by gel filtration, ion-exchange chromatography, and liposome incorporation. The size of the proteoglycans appeared to be directly proportional to the size of their heparan-sulfate chains, larger proteoglycans yielding larger chains. The chondroitin sulfate chains, in contrast, showed no size heterogeneity. Digestion of 125I-labeled proteoglycans with heparitin-sulfate lyase and chondroitin ABC lyase yielded core proteins of approximately 93 kDa, approximately 85 kDa and approximately 38 kDa. Comparison with single enzyme digestions identified the 93-kDa and 85-kDa cores as components of hybrid proteoglycans that carried both heparan-sulfate and chondroitin-sulfate chains. Immunoblotting indicated that the 93-kDa and 85-kDa cores shared the epitope defined by monoclonal antibody 281-2. The 38-kDa core, in contrast, carried only heparan-sulfate chains and lacked the 281-2 epitope. Preparations enriched in heparan sulfate or in heparan-sulfate/chondroitin-sulfate hybrid proteoglycans were obtained by N-desulfation and ion-exchange chromatography. Hybrid proteoglycans accounting for the bulk of the chondroitin-sulfate and nearly half of the heparan-sulfate residues of the proteoglycans showed a similar polydispersity of heparan-sulfate chain sizes as found in proteoglycans that carried only, or predominantly, heparan-sulfate chains. These hybrids contained heparan-sulfate and chondroitin-sulfate chains in similar molar amounts. Analysis of 125I-labeled proteoglycans suggested that typical hybrid proteoglycans were composed of a 85-kDa core protein that carries a single chondroitin-sulfate chain and a single heparan-sulfate chain of variable length. A minority of hybrids seemed characterized by the variant, but possibly structurally related, 93-kDa core protein. The other half of the hydrophobic proteoglycans were composed of the 38-kDa core and carried only heparan-sulfate chains. The significance of the co-existence of hybrid and heparan-sulfate proteoglycans at the cell surface and possible relationships between the proteoglycans need to be further clarified.

Organisation of the chloroplast genome of kiwifruit (Actinidia deliciosa)

A restriction map of the chloroplast genome has been determined for kiwifruit, Actinidia deliciosa. Single and multiple enzyme digests of kiwifruit chloroplast DNA were hybridised to a set of Brassica chloroplast probes, and the kiwifruit bands aligned with the known Brassica map. The chloroplast DNA of kiwifruit is typical of the majority of angiosperm chloroplast genomes; it is 160 kb in size, contains a 15–34 kb inverted repeat, and its gene content and gene order are similar to those of the Brassica chloroplast genome.

Chloroplast genome characterization in the red alga Griffithsia pacifica

It has been suggested that cyanobacteria served as the ancestors for rhodophytic algae whose chloroplasts contain chlorophyll a and phycobilins, and that a rodophyte served as the plastid source for chromophytic plants that contain chlorophylls a and c. Although organellar DNA has been used to assess phylogenetic relatedness among terrestrial plants and green algae whose chloroplasts contain chlorophylls a and b, few data are presently available on the molecular profile of plastid DNA in chromophytes or rhodophytes. In this study the chloroplast genome of the rhodophytic, filamentous alga Griffithsia pacifica has been characterized. DNA was purified from isolated chloroplasts using protease k treatment and sodium dodecyl sulfate lysis followed by density centrifugation in Hoechst-33258 dye-CsCl gradients. Single and double restriction enzyme digests demonstrate that the DNA prepared from purified chloroplasts has a genome size of about 178 kilobase pairs (kb). A restriction map of this chloroplast genome demonstrates that it is circular and, unlike the chloroplast DNA (cpDNA) in most other plants, contains only a single ribosomal DNA operon. DNA was also purified from the mitochondria that co-isolated with chloroplasts. Mitochondrial DNA consists of molecules that range in size from 27 to 350 kb based on restriction endonuclease digestion and electron microscopic analysis.

STEROID 21-HYDROXYLASE GENE ANALYSIS IN CONGENITAL ADRENAL HYPERPLASIA

Congenital adrenal hyperplasia (CAH) is usually due to an autosomal recessive defect in 21-hydroxylation of steroid hormones. The human gene for 21-hydroxylation, P450c21B, is closely linked to its inactive homologue, P450c21A. We have cloned a human P450c21 cDNA and used it to determine the restriction patterns of 51 members of 10 different families, each with 2 or more CAH cases. Following digestion of genomic DNA with EcoRI or TagI, patterns reported by others to indicate P450c21B gene deletions (absent 12- or 3.7-kb fragments) were seen for 6/20 CAH and 6/20 normal P450c21B alleles. Following digestion with Bg1II, EcoRI, KpnI, TaqI, or XbaI normal P450c21B gene patterns were seen in all CAH samples with at least 2 enzymes. Linkage analysis indicates the P450c21 alleles co-segregate with CAH (no recombinants seen in 15 informative meioses). We conclude 1) P450c21B gene “deletions” detected by others using single enzyme digestions may, in some cases, represent gene conversions, polymorphisms or unequal crossover products rather than simple deletions and 2) while prenatal diagnosis of CAH is feasible by linkage analysis in 8/10 families studied to avoid errors the patterns of all family members should be studied before inferring the CAH status of the fetus from restriction patterns.