Single Restriction Site Research Articles

Construction and identification of an infectious clone for CDV-3 strain of canine distemper virus

In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 10⁷·⁶⁶⁷ TCID₅₀/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 10⁶·⁶⁶⁷ TCID₅₀/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.

Advancing DNA Steganography with Incorporation of Randomness.

DNA has become a promising candidate as a future data storage medium; this makes DNA steganography indispensable in DNA data security. PCR primers are conventional secret keys in DNA steganography. Brute force testing of different primers will be extremely time consuming, and practically unaffordable when high‐throughput sequencing is used. However, the encrypted information can be sequenced and read once the primers are intercepted. A new steganography approach is needed to make the DNA‐encoded information safer, if not unhackable. Mixing information‐carrying DNA with a partially degenerated DNA library containing single or multiple restriction sites, we have built an additional protective layer that can be removed by desired restriction enzymes as secondary secret keys. As PCR is inevitable for reading DNA‐encrypted information, heating will cause reshuffling and generate endonuclease‐resistant mismatched duplexes, especially for DNA with high sequence diversity. Consequently, with the incorporation of randomness, DNA steganography possesses both quantum key distribution (QKD)‐like function for detecting PCR by an interceptor and a self‐destructive property. It is noteworthy that the background noise generated through the protective layer is independent from any sequencing technology including Sanger and high‐throughput sequencing. With a DNA ink incorporating the steganography, we have shown that the authenticity of a piece of writing can be confirmed only by authorized persons with knowledge of all embedded keys.

Genome-Wide Association Study and Fine Mapping Reveals Candidate Genes for Birth Weight of Yorkshire and Landrace Pigs.

Birth weight of pigs is an important economic factor in the livestock industry. The identification of the genes and variants that underlie birth weight is of great importance. In this study, we integrated two genotyping methods, single nucleotide polymorphism (SNP) chip analysis and restriction site associated DNA sequencing (RAD-seq) to genotype genome-wide SNPs. In total, 45,175 and 139,634 SNPs were detected with the SNP chip and RAD-seq, respectively. The genome-wide association study (GWAS) of the combined SNP panels identified two significant loci located at chr1: 97,745,041 and chr4: 112,031,589, that explained 6.36% and 4.25% of the phenotypic variance respectively. To reduce interval containing causal variants, we imputed sequence-level SNPs in the GWAS identified regions and fine-mapped the causative variants into two narrower genomic intervals: a ∼100 kb interval containing 71 SNPs and a broader ∼870 kb interval with 432 SNPs. This fine-mapping highlighted four promising candidate genes, SKOR2, SMAD2, VAV3, and NTNG1. Additionally, the functional genes, SLC25A24, PRMT6 and STXBP3, are also located near the fine-mapping region. These results suggest that these candidate genes may have contribute substantially to the birth weight of pigs.

DEVELOPMENT OF TERMITOMYCES PROTOCLONES AND CHARACTERIZATIONS THROUGH PCR BASED ISSR AND RFLP OF RRNA-ITS GENE

A total of five protoclones were successfully cultured on PDA medium out of regenerated twenty two colonies of Termitomyces protoplast and further studied. Liquid MYG grown mycelial tissue is used for protoplast isolation by enzymatic digestion in a mixture containing Lysing enzyme 2% and Cellulase R10 2% in 0.6 M mannitol. The incubation conditions like temperature, shaking and time were standardized at 24oC with shaking 60 rpm for 10 hours, respectively for healthy protoplasts liberation. The purified protoplasts showed 31.60±9.31% regeneration on specific medium and 77.12±2.72% viability by FDA test. Four ISSR primers were used in this study with a total of 27 reproducible bands with mean value 6.75 and they showed similar banding pattern in all with zero percent polymorphism ranged from 280 bp - 2700 bp. The amplified rRNA-ITS gene showed ~ 600 bp size in gel and found a single restriction site for enzyme HaeIII in all the protoclones and parent with similar fragment size in all.

Haplotype Diversity at Sub1 Locus and Allelic Distribution Among Rice Varieties of Tide and Flood Prone Areas of South-East Asia

Single nucleotide polymorphisms and restriction digestion-based haplotype variations among 160 flood prone rice varieties were analyzed with enzymes Alu I and Cac8 I to generate polymorphisms at Sub1A and Sub1C loci (conferring submergence tolerance), respectively. Haplotype associated with phenotype was used to study the haplotype variations at Sub1A and Sub1C loci and to determine their functional influence on submergence tolerance and stem elongation. Three patterns at Sub1A locus, Sub1A0 (null allele), Sub1A1 (does not cut) and Sub1A2 (one SNP), and four patterns at Sub1C locus, Sub1C1, Sub1C2, Sub1C3 and Sub1C4, were generated. Both tolerant Sub1A1 and intolerant Sub1A2 had the same length, but the difference was presence of a restriction site in the Sub1A2, but absent at the Sub1A1. Further, two types of polymorphism were detected at the Sub1C, one included major length polymorphisms (165, 170 and 175bp) and the other was a single restriction site at different position. Eight haplotypes (different combinations of the two loci), A1C1, A1C2, A1C4, A2C2, A2C4, A0C2, A0C3 and A0C4, were detected among 160 varieties. Haplotype A1C1 was comparatively more related to haplotypes A1C2 and A1C4, having the same Sub1A allele, and these haplotypes were found only in Bangladeshi, Sri Lankan and Indian varieties. Most tolerant varieties in A1C1 haplotype showed slow elongation, having tolerant specific Sub1A1 and Sub1C1 alleles. Further, the varieties Madabaru and Kottamali (A2C2) also showed moderate level of tolerance without Sub1A1 allele. These varieties were different with FR13A and also suspected to carry different novel tolerant genes at other loci. These materials could be used for hybridization with Sub1 varieties for pyramiding additional tolerant specific alleles into a single genotype for improving submergence tolerance in rice.

The use of the replication region of plasmid pRS7 from Oenococcus oeni as a putative tool to generate cloning vectors for lactic acid bacteria

A chimeric plasmid, pRS7Rep (6.1 kb), was constructed using the replication region of pRS7, a large plasmid from Oenococcus oeni, and pEM64, a plasmid derived from pIJ2925 and containing a gene for resistance to chloramphenicol. pRS7Rep is a shuttle vector that replicates in Escherichia coli using its pIJ2925 component and in lactic acid bacteria (LAB) using the replication region of pRS7. High levels of transformants per µg of DNA were obtained by electroporation of pRS7Rep into Pediococcus acidilactici (1.5 × 107), Lactobacillus plantarum (5.7 × 105), Lactobacillus casei (2.3 × 105), Leuconostoc citreum (2.7 × 105), and Enterococcus faecalis (2.4 × 105). A preliminary optimisation of the technical conditions of electrotransformation showed that P. acidilactici and L. plantarum are better transformed at a later exponential phase of growth, whereas L. casei requires the early exponential phase for better electrotransformation efficiency. pRS7Rep contains single restriction sites useful for cloning purposes, BamHI, XbaI, SalI, HincII, SphI and PstI, and was maintained at an acceptable rate (>50%) over 100 generations without selective pressure in L. plantarum, but was less stable in L. casei and P. acidilactici. The ability of pRS7Rep to accept and express other genes was assessed. To the best of our knowledge, this is the first time that the replication region of a plasmid from O. oeni has been used to generate a cloning vector.

Bedrest increases burden of mitochondrial DNA deletions in human muscle (956.1)

We explored whether muscle disuse (i.e. bed rest) contributes to mitochondrial (mt)DNA deletions in skeletal muscle. mtDNA deletions have been associated with various degenerative processes (e.g. sarcopenia) and sedentary behavior. Human muscle tissue from a small sample (n=3) taken before and after a 28‐day bed rest were subjected to mutational analysis using, a restriction digestion / long range digital PCR approach. Total DNA was digested with SnaBI, an enzyme with a single restriction site located in an area commonly removed in deleted mtDNA molecules. DNA was exposed to long‐range PCR capable of amplifying most of the mitochondrial genome. Restriction digestion rendered mtDNA un‐amplifiable, except for molecules with deletions. The actual number of molecules with deletions and types thereof were determined using highly diluted DNA samples, reaching single molecule per reaction, in multiple independent PCR reactions, so that molecules could be directly counted (“digital PCR”). We found an increase in the fraction of molecules with mtDNA deletions after bedrest, relative to intact wild type molecules. This finding suggests that disuse results in skeletal muscle mtDNA deletions. Further studies are needed to determine the association of these deletions with physiological and metabolic changes seen with disuse or sedentary behavior.Grant Funding Source: Supported by Ellison Medical Foundation, NSBRI‐NPFR00301, GCRC M01 RR00

A Novel Approach for Secretion of Heterologous Proteins with Correct N-Terminal Processing by Using α-Factor Pre Sequence in Pichia pastoris

Numerous proteins have been secreted in P. pastoris by fusing the target gene with α-factor pre-pro sequence at Kex2 endopeptidase cleavage site. However, in some instances the product cannot be correctly processed due to aberrant cleavage by Kex2 endopeptidase such as aprotinin. In this study, an aprotinin gene was cloned into pPIC9K at the signal peptidase cleavage site through a single NheI restriction site designed at the 3'end of the α-factor signal sequence preregion, and transformed into GS115 host cell. By G418 resistance and ELISA assay, a high-yield recombinant was selected. After fed-batch cultivation in a 7-L bioreactor, the product was efficiently secreted into culture medium and accumulated up to ~4.7 mg L⁻¹. MALDI-TOF/MS and N-terminal analyses confirmed its authenticity. Thus, a novel cloning strategy for secretion of aprotinin with correct N-terminal processing in P. pastoris has been developed which can be potentially applied to other proteins.

Digestion-after-shuffling: A method for combinatorial library construction

We have developed a convenient method for family shuffling of amino acid sequences, termed digestion-after-shuffling. After DNA shuffling of homologous genes, plasmid mixture is extracted from a library and used for several double digestions with restriction enzymes. For each double digestion, two restriction enzymes are selected, corresponding to the single restriction sites of different parental genes. After digestions, fragments with expected sizes are obtained by gel purification and religated to construct recombinant plasmids. Thus, the obtained genes should be chimeras and have at least two restriction sites originating from different parental sequences.

First Report of the Peach Brown Rot Fungus Monilinia fructicola Resistant to Demethylation Inhibitor Fungicides in New Jersey.

Reduced sensitivity and resistance of Monilinia fructicola to demethylation inhibitors (DMIs; fungicide group 3) have been previously found in stone fruit orchards in Georgia, South Carolina, Ohio, and New York (2). Resistance development is a major concern because of the importance of DMIs for brown rot management. Eleven single-spore isolates, originally collected during 2006 from separate commercial peach (Prunus persica) orchards in southern New Jersey, were removed from cold storage (5°C) in early 2008 and examined in vitro for resistance to the DMI propiconazole (Orbit 3.6EC; Syngenta Crop Protection, Inc., Greensboro, NC). After 19 months at 5°C, isolate 7 was inhibited 53.4% in growth on potato dextrose agar (PDA) amended at the discretionary dose of 0.3 μg/ml propiconazole; inhibition of the remaining isolates ranged from 81.4 to 100%. Inhibition values were based on two replications of eight colonies per isolate performed after incubation at 25°C for 4 days. Because of the previously reported relationship between duration of cold storage and propiconazole sensitivity, isolate 7 was tentatively deemed resistant (1). To confirm the in vitro results, isolates were grown at 25°C for 7 days on cellophane over PDA. Genomic DNA was isolated from mycelium with the DNeasy Plant Mini Kit (Qiagen, Inc., Valencia, CA). PCR with primers INS65-F and INS65-R was conducted on a GeneAmp thermal cycler (Applied Biosystems, Inc., Foster City, CA) as described previously to amplify a 65-bp region named 'Mona' associated with DMI resistance (2). PCR products were separated via electrophoresis on 0.8% agarose gel. The primers amplified a 376-bp fragment from isolate 7 and a 311-bp fragment from all other isolates, thus indicating the presence of Mona in isolate 7. Restriction fragment length polymorphism analysis using the BsrBI enzyme, specific to a single restriction site within Mona, was conducted on the amplified fragments from all isolates. Electrophoresis results showed digestion of the 376-bp fragment from isolate 7 into 140-bp and 236-bp fragments, thereby confirming the presence of Mona; none of the 311-bp fragments from the remaining isolates were cut by BsrBI. Although economic loss from brown rot has not been reported in New Jersey, these results show that propiconazole-resistant strains have been detected since 2006 and it is most likely that resistant strains of the pathogen are still present in commercial peach orchards. To combat this risk, current brown rot control recommendations are incorporating quinone outside inhibitors (QoIs; fungicide group 11) and carboxamides (fungicide group 7) into control programs as a resistance management strategy. More extensive sampling is planned to ascertain the prevalence and location of resistant strains.

Prion protein detection via direct immuno-quantitative real-time PCR

We describe a simple and robust assay for the quantitative detection of prions using immuno-quantitative real-time PCR (iQ-RT-PCR) made possible by a direct conjugate of a prion-specific antibody (ICSM35) and a synthetic 99-bp DNA tail. The DNA tail was engineered to include a single ScrFI restriction site, which enabled subsequent quantification of restricted DNA tails using real-time PCR. The assay was tested with scrapie prions bound to polyvinylidene difluoride membranes and to 96-well plates coated with a capturing antibody from a commercially available immuno-based assay (TeSeE). The iQ-RT-PCR assay had a detection limit corresponding to 2.32 × 10 2 prion epitopes, which represented a 1000-fold increase in detection sensitivity over the commercial assay. Detection of prions from diluted scrapie-positive brain homogenate bound to membranes was linear over a range of 1.06 × 10 4 to 3.24 × 10 2 epitopes ( R 2 = 0.92). Given its sensitivity and versatility, the present assay has potential to enable rapid and reliable detection of agents causing transmissible spongiform encephalopathies.

Intra-specific rDNA-ITS restriction site variation and an improved protocol to distinguish species and hybrids in the Daphnia longispina complex

A collaborative research effort was undertaken to evaluate the robustness of a recently developed genetic tool for species identification of members in the morphologically variable Daphnia longispina species complex. This genetic method, based on restriction fragment length polymorphism (RFLP) of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA (rDNA) with restriction enzymes Mwo I and Sau96 I [Billiones et al., 2004. Hydrobiologia 526: 43–53], was applied to many different European populations. Results were compared with two or more independently obtained characters (morphology, allozymes, mitochondrial DNA (mtDNA), or cloned rDNA-ITS sequences). Individuals of most taxa were readily identified, but unexpected ITS-RFLP patterns were found in many individuals indicated by other markers to be D. galeata or one of its hybrids. Among 43 investigated D. galeata populations (902 specimen analysed by ITS-RFLP), deviant RFLP fragment patterns occurred in 26 (i.e., more than half) of the populations. The deviant patterns could be attributed to the loss of one single restriction site in the ITS2 region. This loss made the distinction of D. galeata from other species unreliable, and F1 hybrids could not be identified. Future users should be aware of this shortcoming of the Billions et al. [2004. Hydrobiologia 526: 43–53] protocol. As a solution to this problem, we present an improved genetic identification protocol based on a simple double digestion of the rDNA-ITS region with the restriction enzymes BsrB I and EagI. Sequence analyses of rDNA-ITS clones and preliminary testing indicate that the new protocol is unaffected by the rDNA variation which troubled the Mwo I/Sau96 I protocol. Further, the new protocol identifies all European species of the D. longispina complex, as well as their F1 hybrids. However, a wider screening is required to verify its general utility for all species, since yet unknown variation may occur.

Identification of Phytophthora Isolates to Species Level Using Restriction Fragment Length Polymorphism Analysis of a Polymerase Chain Reaction-Amplified Region of Mitochondrial DNA

ABSTRACT Polymerase chain reaction primers spanning the mitochondrially encoded coxI and II genes have been identified that were capable of amplifying target DNA from all 152 isolates of 31 species in the genus Phytophthora that were tested. Digestion of the amplicons with restriction enzymes generated species-specific restriction fragment length polymorphism banding profiles that were effective for isolate classification to a species level. Of the 24 species in which multiple isolates were examined, intraspecific polymorphisms were not observed for 16 species, while 5 species exhibited limited intraspecific polymorphism that could be explained by the addition/loss of a single restriction site. Intraspecific polymorphisms were observed for P. megakarya, P. megasperma, and P. syringae; however, these differences may be a reflection of the variation that exists in these species as reported in the literature. Although digestion with AluI alone could differentiate most species tested, single digests with a total of four restriction enzymes were used in this investigation to enhance the accuracy of the technique and minimize the effect of intraspecific variability on correct isolate identification. The use of the computer program BioNumerics simplified data analysis and identification of isolates. Successful template amplification was obtained with DNA recovered from hyphae using a boiling miniprep procedure, thereby reducing the time and materials needed for conducting this analysis.

A Superfamily of Voltage-gated Sodium Channels in Bacteria

NaChBac, a six-alpha-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na(+)-selective channel. As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na(+) channels (Na(V)PZ from Paracoccus zeaxanthinifaciens and Na(V)SP from Silicibacter pomeroyi). Na(V)PZ and Na(V)SP share approximately 40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both Na(V)PZ and Na(V)SP were Na(+)-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-alpha-helical transmembrane-spanning subunits constitute a widely distributed superfamily (Na(V)Bac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of Na(V)Bac structure and function of similarity and dissimilarity among Na(V)Bac proteins. Thus, the Na(V)Bac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.

Efficient expression and secretion of recombinant hirudin III in E. coli using the l-asparaginase II signal sequence

One of the hirudin variants HV3 was efficiently expressed in Escherichia coli using the l-asparaginase II signal sequence and the product was secreted into the culture medium. For the secretory manufacture of HV3, the l-asparaginase II signal sequence containing a single NheI restriction site at its 3 ′ end was designed using the degenerate codons and PCR-amplified from E. coli chromosomal DNA. The synthetic HV3 coding sequence was fused to the signal sequence in-frame by its 5 ′ NheI restriction site. The above signal-HV3 fusion gene was inserted into an expression vector pTA, which was derived from pkk223-3 such that its expression was under the control of the tac promotor. The resulting HV3 secretion expression vector pTASH thus constructed was introduced into an E. coli host cell AS1.357 with high l-asparaginase II producing level. After inducing with IPTG, the expression product was efficiently secreted into the culture medium and shake-flask culturing gave a yield of approximately 5×10 5 ATU/L (∼ 60 mg/ L ). The secreted HV3 was easily purified from culture supernatant using ultrafiltration, ion-exchange column chromatography, and FPLC reverse-phase chromatography. The purified rHV3 from the culture supernatant had the expected N-terminal amino sequence and strong antithrombin activity, suggesting that the signal sequence was completely removed and the product was processed accurately during the secretion process.

Identification of Toxin and Fish Species in Cooked Fish Liver Implicated in Food Poisoning

ABSTRACT: The cooked fish liver retained by the victims was assayed for toxicity and mitochondrial DNA. Meanwhile, 8 live specimens of puffer Takifugu niphobles were also assayed. The toxicity of cooked fish liver was 280 ± 20 mouse units per gram (MU/g). All specimens of T. niphobles showed high toxicity (more than 850 MU/g) in the liver. The toxin from cooked fish liver and liver of T. niphobles was identified to be tetrodotoxin. The cooked fish liver and fresh liver of T. niphobles showed the same sequence genotype and the same single restriction site for Bsa I. Therefore, the species of cooked fish liver was suggested as T. niphobles.

Identification of Tetrodotoxin and Fish Species in a Dried Dressed Fish Fillet Implicated in Food Poisoning

There were five victims of neurotoxic food poisoning from a dried dressed fish fillet in Changhua County, Taiwan, in February 2000. The toxicity of the dried dressed fish fillets was 243 mouse units per g according to a tetrodotoxin bioassay. The partially purified toxin was identified as tetrodotoxin and anhydrotetrodotoxin. The sequence of the 376-nucleotide region in the cytochrome b gene of the mitochondrial DNA exhibited the same genotype as that of the toxic puffer fish Lagocephalus lunaris. The same single restriction site for HinfI was found in the polymerase chain reaction (PCR) products from the dried dressed fish fillet and the muscle of L. lunaris, yielding two DNA fragments of 170 and 206 bp. However, no restriction site for HinfI was found in the PCR products from other toxic puffer fishes, including Takifugu niphobles, Takifugu oblongus, and Takifugu rubripes. Therefore, the species of the dried dressed fish fillet was identified as L. lunaris and its causative agent was identified as tetrodotoxin.

Production in mammalian cells of chimeric human/sea urchin procollagen molecules displaying distinct versions of the minor triple helix.

One of the mechanisms involved in the regulation of the fibril diameter is the retention of the N-propeptide. In sea urchin embryo, thin collagen fibrils harbor numerous extensions at their surface, which we have suggested correspond to the large N-propeptide of the 2alpha collagen chain. To investigate the function of the N-propeptide during fibrillogenesis, we engineered constructs coding for the globular region of the 2alpha N-propeptide. To obtain homotrimeric molecules, the N-telopeptide, the central triple helix and the C-propeptide of the 2alpha chain were replaced by human domains of the proalpha1(I) chain. A single restriction site allowed insertion of distinct versions of the minor triple helix of the N-propeptide. Several human cell lines were transfected, and with one of them we were able to produce intact homotrimeric procollagen molecules. Rotary shadowing of these purified molecules indicates the presence of three large 2alpha N-propeptides that are similar to the extensions present at the surface of the sea urchin thin fibrils. This cassette-vector will be useful in determining the respective contributions of the globular and minor triple helical domains of the N-propeptide in the regulation of fibril diameter.

A New Molecular Phylogenetic Hypothesis for the Evolution of Freshwater Eels

Phylogenetic analysis of a segment of the mitochondrial 16S rDNA of eight Anguilla species from the Indo–Pacific region and from the North Atlantic revealed that the genus Anguilla appears to be surprisingly young, based upon the small observed maximum genetic distance of 4.8% and the high degree of morphological similarity among the species. The placement of A. marmorata as the most ancestral lineage suggests that the genus is likely to have originated in the Indo–Malayian region, from which it quickly spread. Two Pacific species, A. obscura and A. japonica, branched next. A. japonica was placed as sister group to all remaining species, which formed three clades: the first comprising A. australis, the second A. reinhardti and A. mossambica, and the third A. anguilla and A. rostrata. All analyzed specimens of A. rostrata originating from southern New Jersey to Nova Scotia had identical mitotypes, while five mitochondrial genotypes were found in Europe differing by zero to two substitutions. The two Atlantic eel species are very closely related; all surveyed specimens of A. anguilla differ by three to five substitutions from their American allies, corroborating the existence of two distinct biological species. This was also confirmed by restriction analysis of a 350-bp segment of the cytochrome b, in which American specimens were distinct in sharing a single diagnostic restriction site of HinfI. Our results suggest little to no gene flow between the two nominal Atlantic eel species.

Atypical beta(s) haplotypes are generated by diverse genetic mechanisms.

The majority of the chromosomes with the beta(S) gene have one of the five common haplotypes, designated as Benin, Bantu, Senegal, Cameroon, and Arab-Indian haplotypes. However, in every large series of sickle cell patients, 5-10% of the chromosomes have less common haplotypes, usually referred to as "atypical" haplotypes. In order to explore the genetic mechanisms that could generate these atypical haplotypes, we extended our analysis to other rarely studied polymorphic markers of the beta(S)-gene cluster, in a total of 40 chromosomes with uncommon haplotypes from Brazil and Cameroon. The following polymorphisms were examined: seven restriction site polymorphisms of the epsilongammadeltabeta-cluster, the pre-(G)gamma framework sequence including the 6-bp deletion/insertion pattern, HS-2 LCR (AT)xR(AT)y and pre-beta (AT)xTy repeat motifs, the GC/TT polymorphism at -1105-1106 of (G)gamma-globin gene, the C/T polymorphism at -551 of the beta-globin gene, and the intragenic beta-globin gene framework. Among the Brazilian subjects, the most common atypical structure (7/16) was a Bantu 3'-subhaplotype associated with different 5'-sequences, while in two chromosomes a Benin 3'-subhaplotype was associated with two different 5'-subhaplotypes. A hybrid Benin/Bantu configuration was also observed. In three chromosomes, the atypical haplotype differed from the typical one by the change of a single restriction site. In 2/134 chromosomes identified as having a typical Bantu RFLP-haplotype, a discrepant LCR repeat sequence was observed, probably owing to a crossover 5' to the epsilon-gene. Among 80 beta(S) chromosomes from Cameroon, 22 were associated with an atypical haplotype. The most common structure was represented by a Benin haplotype (from the LCR to the beta-gene) with a non-Benin segment 3' to the beta-globin gene. In two cases a Bantu LCR was associated with a Benin haplotype and a non-Benin segment 3' to the beta-globin gene. In three other cases, a more complex structure was observed that can be considered as a hybrid of Benin, Bantu, Senegal, or other chromosomes was observed. These data suggest that the atypical beta(S) haplotypes are not uncommon in America and in Africa. These haplotypes are probably generated by a variety of genetic mechanisms including (a) isolated nucleotide changes in one of the polymorphic restriction sites, (b) simple and double crossovers between two typical beta(S) haplotypes or much more frequently between a typical beta(S) haplotype and a different beta(A)-associated haplotype that was present in the population, and (c) gene conversions.