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PubMed1998

Orthologous DNA sequence variation among 5S ribosomal RNA gene spacer sequences on homoeologous chromosomes 1B, 1D, and 1R of wheat and rye.

5S ribosomal gene spacer sequences from the short-spacer arrays of wheat and rye were isolated by PCR. The 29 new DNA sequences displayed noticeable heterogeneity at scattered positions. Nevertheless, based on shared DNA sequence polymorphisms, sequence alignment clearly classified the sequences into three groups. Group-specific primer sets were designed to allow chromosomal assignment by PCR on nullitetrasomic wheat stocks, as well as on wheat-rye translocation and addition lines. The three groups were assigned to orthologous loci 5S-Rrna-B1, 5S-Rrna-D1, and 5S-Rrna-R1 on homoeologous chromosomes 1B, 1D, and 1R, respectively. Hence, group-specific DNA sequence variation could be related to fixed orthologous DNA sequence variation between 5S rRNA multigene families on the homoeologous group 1 chromosomes. In addition, members of the three groups showed fixed orthologous length polymorphism. Four sequenced 5S-Rrna-B1 units, however, had a duplication in the gene encoding region and are probably representatives of a nontranscribed subfamily of 5S rDNA repeating units. The observed chromosome-specific polymorphisms among sequences belonging to a multigene family with thousands of copies suggests that this type of polymorphism may exist in many genes and gene families in polyploid wheats. The implication of this finding in relation to the construction of molecular tools for wheat-genome analysis and manipulation is discussed.

Base Sequence↗
PubMed1990

Sequence analyses of the 3' genome end and NP gene of human parainfluenza type 2 virus: sequence variation of the gene-starting signal and the conserved 3' end.

We cloned and determined the nucleotide sequences of cDNAs against nucleocapsid protein (NP) mRNA and the genomic RNA of human parainfluenza type 2 virus (PIV-2). The 3' terminal region of genomic RNA was compared among PIV-2, mumps virus (MuV), Newcastle disease virus (NDV), measles virus (MV), PIV-3, bovine parainfluenza type 3 virus (BPIV-3), Sendai virus (SV), and vesicular stomatitis virus (VSV), and an extensive sequence homology was observed between PIV-2 and MuV. Although no significant sequence relatedness was observed between PIV-2 and other viruses, the terminal four nucleotides were identical in the viruses compared, implying a specific role of these nucleotides on the replication of paramyxoviruses. A primer extension analysis elucidated the major NP mRNA initiation site with the sequence UCUAAGCC, which showed a moderate homology with the gene-starting consensus sequences of other paramyxoviruses. On the other hand, the NP mRNA was terminated at the nucleotide stretch AAAUUCUUUUU, and this sequence was conserved in all the PIV-2 genes, indicating that the oligonucleotides will form a part of the gene attenuation signal of PIV-2. Comparisons of NP protein sequence indicated a possible subgrouping of the paramyxoviruses into two groups, one of which is a group including PIV-2, PIV-4, MuV, and NDV, and another is a group including PIV-3, BPIV-3, and SV. This result supports an idea from our previous studies using polyclonal and monoclonal antibodies. Furthermore, our data indicated that the PIV-2 NP protein sequence was more closely related to MV and CDV than to other parainfluenza viruses, PIV-3 and SV.

Amino Acid Sequence↗
PubMed1999

Identification of mammalian mitochondrial ribosomal proteins (MRPs) by N-terminal sequencing of purified bovine MRPs and comparison to data bank sequences: the large subribosomal particle.

Bovine mitochondrial ribosomes are presented as a model system for mammalian mitochondrial ribosomes. An alternative system for identifying individual bovine mitochondrial ribosomal proteins (MRPs) by RP-HPLC is described. To identify and to characterize individual MRPs proteins were purified from bovine liver, separated by RP-HPLC, and identified by 2D PAGE techniques and immunoblotting. Molecular masses of individual MRPs were determined. Selected proteins were subjected to N-terminal amino acid sequencing. The peptide sequences obtained were used to screen different databases to identify several corresponding MRP sequences from human, mouse, rat, and yeast. Signal sequences for mitochondrial import were postulated by comparison of the bovine mature N-termini determined by amino acid sequencing with the deduced mammalian MRP sequences. Significant sequence similarities of these new MRPs to known r-proteins from other sources, e.g., E. coli, were detected only for two of the four MRP families presented. This finding suggests that mammalian mitochondrial ribosomes contain several novel proteins. Amino acid sequence information for all of the bovine MRPs will prove invaluable for assigning functions to their genes, which would otherwise remain unknown.

Amino Acid Sequence↗
PubMed1992

Sheep 6-phosphogluconate dehydrogenase. Revised protein sequence based upon the sequences of cDNA clones obtained with the polymerase chain reaction.

Sheep liver 6-phosphogluconate dehydrogenase (6-PGDH) is an enzyme of the pentose phosphate pathway. Evidence has appeared which suggests that the 6-PGDH protein sequence determined previously by direct analysis of the protein isolated from ovine liver is incorrect. Determining the enzyme's DNA sequence was considered to be the best way of solving the problem. In the first instance, a degenerate forward and a degenerate reverse primer were designed on the basis of the known protein sequence, and a partial-length cDNA clone was isolated from total sheep liver cDNA using the polymerase chain reaction. The clone encoded the expected part of the protein sequence. The clone was unsuccessfully used as a prime-cut probe to screen a sheep liver library and a bovine heart library. As a result, the polymerase chain reaction was utilized again to successfully generate a family of overlapping cDNA clones encoding a mature protein of 482 amino acids. The mature protein sequence encoded by the cDNA differs significantly from the sequence derived by direct analysis of the protein, but on closer examination the fundamental difference is caused by the incorrect placement of three enzyme fragments obtained by cyanogen bromide cleavage during the direct sequence analysis of the protein. Placing the fragments in the correct order results in the two sequences being virtually identical except for some minor amino acid changes between the amide and acid forms, and a small number of deletions and insertions.

Amino Acid Sequence↗
PubMed2000

Development and characterization of expressed sequence tags for the turkey (Meleagris gallopavo) genome and comparative sequence analysis with other birds.

Twenty-one randomly selected clones from a turkey (Meleagris gallopavo) pituitary complementary DNA (cDNA) library were sequenced to develop expressed sequence tags (ESTs) for this economically important avian species whose genome is among the least understood. Primers specific for the ESTs were used to produce amplicons from the genomic DNA of turkey, chicken (Gallus gallus), guinea fowl (Numidia meleagris), pigeon (Columba domestica), and quail (Corturnix japonica). The amplicons were sequenced and analyzed for sequence variation within- and similarity among-species and with GenBank database sequences. The proportion of shared bases between the turkey sequence and the consensus sequence from each of the other species ranged from 72% to 93% between turkey and pigeon and quail and between turkey and chicken, respectively. The total number of single nucleotide polymorphisms (SNPs) observed ranged from 3 in quail to 18 in chicken out of 4898 and 5265 bases analyzed, respectively. The most frequent nucleotide variation observed was a C-->T transition. Linkage analysis of one such SNP in the backcross progeny of the East Lansing reference DNA panel, localized TUS0005, the chicken sequence derived from primers specific for turkey TUT2E EST, to chromosome 4. The ESTs reported, as well as the SNPs may provide a useful resource for ongoing efforts to develop high utility genome maps for the turkey and chicken. The primers described can also be used as a tool in future investigations directed at further understanding the biology of the guinea fowl, pigeon and quail and their relatedness to the turkey.

Animals↗
PubMed1997

Sequence heterogeneity of heron hepatitis B virus genomes determined by full-length DNA amplification and direct sequencing reveals novel and unique features.

So far, only a single heron hepatitis B virus genome (HHBV-4) has been cloned and sequenced. Therefore, neither the significance of its sequence divergence from other avian hepadnaviruses nor the sequence variability of HHBV genomes in general are known. Here we have analysed the sequence heterogeneity of HHBV genome populations in several sera from naturally infected herons. A highly sensitive PCR method for full-length HHBV genome amplification was established which allowed direct sequencing of entire HHBV populations without prior cloning. Sequences of HHBV genomes from four sera were thus obtained which differed from those of HHBV-4 by up to 7%. Some of the divergent nucleotides and the corresponding amino acids of the predicted viral proteins were conserved in all four new HHBV isolates and varied only in HHBV-4. This indicates that the HHBV-4 genome is not in all aspects representative of this class of viruses. Interestingly, a highly conserved ORF upstream of the C-gene present in a position analogous to that of the mammalian hepadnavirus X-gene became apparent in all HHBV genomes. In contrast to the duck hepadnaviruses, the small (sAg-S) instead of the largest (sAg-L) envelope protein of all HHBVs has a myristylation site. These data confirm the significant sequence divergence of HHBV from other avian hepadnaviruses. Moreover, they show that HHBV has low sequence variability and indicate two new and unique features not evident in other avihepadnaviruses: an additional, highly conserved gene and potential myristylation of the sAg-S instead of the sAg-L envelope protein.

Amino Acid Sequence↗
PubMed1995

Characterization of Newcastle disease virus isolates by reverse transcription PCR coupled to direct nucleotide sequencing and development of sequence database for pathotype prediction and molecular epidemiological analysis.

Degenerate oligonucleotide primers were synthesized to amplify nucleotide sequences from portions of the fusion protein and matrix protein genes of Newcastle disease virus (NDV) genomic RNA that could be used diagnostically. These primers were used in a single-tube reverse transcription PCR of NDV genomic RNA coupled to direct nucleotide sequencing of the amplified product to characterize more than 30 NDV isolates. In agreement with previous reports, differences in the fusion protein cleavage sequence that correlated genotypically with virulence among various NDV pathotypes were detected. By using sequences generated from the matrix protein gene coding for the nuclear localization signal, lentogenic viruses were again grouped phylogenetically separate from other pathotypes. These techniques were applied to compare neurotropic velogenic viruses isolated from an outbreak of Newcastle disease in cormorants and turkeys. Cormorant NDV isolates and an NDV isolate from an infected turkey flock in North Dakota had the fusion protein cleavage sequence 109SRGRRQKRFVG119. The R-for-G substitution at position 110 may be unique for the cormorant-type isolates. Although the amino acid sequences from the fusion protein cleavage site were identical, nucleotide sequence data correlate the outbreak in turkeys to a cormorant virus isolate from Minnesota and not to a cormorant virus isolate from Michigan. On the basis of sequence information, the cormorant isolates are virulent viruses related to isolates of psittacine origin, possibly genotypically distinct from other velogenic NDV isolates. These techniques can be used reliably for Newcastle disease epidemiology and for prediction of pathotypes of NDV isolates without traditional live-bird inoculations.

Amino Acid Sequence↗
PubMed1996

Possible origin of murine AIDS (MAIDS) virus: conversion of an endogenous retroviral p12gag sequence to a MAIDS-inducing sequence by frameshift mutations.

The murine AIDS (MAIDS) virus has a unique sequence in its p12gag region, which is responsible for MAIDS development. A transcript hybridizing with this sequence is expressed in normal C57BL/6 mice. The transcript, designated Edv, has been previously cloned and sequenced (Y. Kubo, Y. Nakagawa, K. Kakimi, H. Matsui, K. Higo, L. Wang, H. Kobayashi, T. Hirama, and A. Ishimoto, J. Gen. Virol. 75:881-888, 1994). Compared with the nucleotide sequence of the helper LP-BM5 ecotropic virus, the pathogenic replication-defective MAIDS virus has a 16-bp deletion and a 1-bp insertion in the 5' and 3' regions of the p12gag sequence, respectively, and the Edv transcript contains only a 3-bp deletion. Therefore, the amino acid sequence of the defective MAIDS virus p12gag region is not homologous to that of the helper virus and the Edv transcript because of the frameshift. To determine whether the amino acid sequence resulting from the frameshift is critical for MAIDS development, we constructed chimeric viruses that contained the p12gag regions of the helper virus and the Edv transcript, respectively, with and without the same frame as the defective MAIDS virus by the artificial frameshift mutations. The mutant viruses with the frameshift mutations induced MAIDS in inoculated mice, but the viruses without the mutations did not. These results suggested that the MAIDS virus was generated by frameshift mutations in the p12gag region of Edv or a related sequence.

Animals↗
PubMed1993

Amino acid sequence of a major human amelogenin protein employing Edman degradation and cDNA sequencing.

The abundant hydrophobic, proline-glutamine, and histidine-rich (over 90%) amelogenins constitute the major class of proteins in forming extracellular enamel matrix. These are thought to play a major role in the structural organization and mineralization of developing enamel. The present report describes the successful sequencing of the major human amelogenin protein, by use of both Edman degradation and cDNA sequencing. When Edman degradation was used, over 75% of the primary structure of the protein was determined. This sequence was supplemented with cDNA sequencing studies, which revealed the predicted sequence of this protein. Together, they provide the complete sequence of an important human enamel protein. The information complements recent studies on bovine and human amelogenin genes. A comparison between the present results and the protein sequences predicted from the corresponding human amelogenin genomic coding regions and that of cDNA sequences of other species is described.

Amelogenin↗
PubMed1996

Identification and characterization of two tandem repeat sequences (TrsB and TrsC) and a retrotransposon (RIRE1) as genome-general sequences in rice.

Three kinds of DNA sequences (here called TrsB, TrsC and RIRE1) have been previously reported to be those repeated in tandem specifically in the wild rice species with FF, CC or EE genome, respectively. To characterize these genome type-specific sequences, we carried out PCR using a pair of primers, which hybridize to a restricted region in the repeating unit sequence and prime DNA synthesis in both directions. Gel electrophoresis and DNA sequencing revealed that PCR using primers for TrsB (or TrsC) amplified the fragments with an integral series of a unit length not only from total DNA of the rice strain with FF (or CC) genome, but also from those of the rice strains with non-FF (or non-CC) genome. TrsB or TrsC was, however, found to be repeated in an extraordinary number of copies in the species with FF or CC genome, respectively, in which the TrsB (or TrsC) sequence has been originally identified. PCR using primers for RIRE1 produced various sizes of fragments from total DNA of the rice strains with EE genome. The fragments, however, showed no progression at interval of the unit length characteristic for tandem repeats. Nucleotide sequencing of the amplified fragments revealed that they were not the sequences repeated in tandem, but were those interspersed as an element having partial homology with the LTR sequences of retrotransposons, Wis-2-1A in wheat and BARE-1 in barley. RIRE1 was present in the rice species with any types of genomes, but in the species with EE genome in an extraordinary number of copies.

Base Sequence↗
PubMed2002

Gene expression profile of human bone marrow stromal cells: high-throughput expressed sequence tag sequencing analysis.

Human bone marrow stromal cells (HBMSC) are pluripotent cells with the potential to differentiate into osteoblasts, chondrocytes, myelosupportive stroma, and marrow adipocytes. We used high-throughput DNA sequencing analysis to generate 4258 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' (97) and 3' (4161) ends of human cDNA clones from a HBMSC cDNA library. Our goal was to obtain tag sequences from the maximum number of possible genes and to deposit them in the publicly accessible database for ESTs (dbEST of the National Center for Biotechnology Information). Comparisons of our EST sequencing data with nonredundant human mRNA and protein databases showed that the ESTs represent 1860 gene clusters. The EST sequencing data analysis showed 60 novel genes found only in this cDNA library after BLAST analysis against 3.0 million ESTs in NCBI's dbEST database. The BLAST search also showed the identified ESTs that have close homology to known genes, which suggests that these may be newly recognized members of known gene families. The gene expression profile of this cell type is revealed by analyzing both the frequency with which a message is encountered and the functional categorization of expressed sequences. Comparing an EST sequence with the human genomic sequence database enables assignment of an EST to a specific chromosomal region (a process called digital gene localization) and often enables immediate partial determination of intron/exon boundaries within the genomic structure. It is expected that high-throughput EST sequencing and data mining analysis will greatly promote our understanding of gene expression in these cells and of growth and development of the skeleton.

Bone Marrow Cells↗
PubMed1981

Chromosomal locations of two DNA segments that flank ribosomal insertion-like sequences in Drosophila: flanking sequences are mobile elements.

We report the chromosomal locations of two repetitive DNA sequences that flank ribosomal insertion-like sequences in Drosophila melanogaster. The chromocentric region of D. melanogaster contains many copies of sequences that are homologous to type 1 ribosomal insertions. These insertion-like elements are interspersed with other DNA segments that we call flanking sequences. Two distinct flanking sequences derived from the same cloned DNA molecule pDmI 101, the HindIII fragments 101E and 101F, were studied. Whole genome Southern blots with DNA from the D. melanogaster stocks Oregon R (P2), gt-1, and gt-X11 showed complex restriction patterns that differed substantially between the three stocks. This and other data show that flanking sequences are members of diverged repetitive sequence families. In situ hybridization to salivary gland chromosomes of gt-1 and gt-X11 showed that both sequences are homologous to the chromocenter and to about 5 to 8 (101E) or 25 to 30(101F) euchromatic sites in each stock. Most, if not all, of these sites differed in gt-1 and gt-X11. Both 101E and 101F are homologous to he chromocenter and very few euchromatic bands in D. simulans, but 101F is homologous to numerous bands in D. mauritiana. We conclude that the flanking sequences represented by 101E and 101F are mobile elements within the genome of Drosophila. These two sequences differ in several structural features from mobile DNA elements previously described in this organism.

Animals↗
PubMedDate not supplied

Hepatic MR imaging: comparison of RARE derived sequences with conventional sequences for detection and characterization of focal liver lesions.

BACKGROUND: We compared two T2-weighted turbo spin echo (TSE) sequences with a T2-weighted conventional SE (CSE) sequence to determine whether sequences derived from rapid acquisition with relaxation enhancement such as TSE could replace CSE for the detection and subsequent characterization of focal liver lesions. METHODS: A total of 55 consecutive patients with 107 liver lesions underwent magnetic resonance imaging examinations at 1.5 Tesla, with a constant imaging protocol. TSE pulse sequences were acquired with eight echo trains (repetition time [TR], 4718 ms; echo time [TE], 90 ms; acquisition time [TA], 4.03 min; and a symmetric k-space ordering scheme) and 11 echo trains (TR, 4200 ms; TE, 140 ms; TA, 4.40 min; and an asymmetric k-space ordering scheme) and compared with CSE (TR, 2300 ms; TE, 45/90 ms; TA, 9.53 min). Images were analyzed qualitatively by scoring image quality and artifacts and counting focal liver lesions by independent reading with consensus obtained for discrepancies. Quantitative analysis was performed by measuring signal-to-noise (S/N), contrast-to-noise (C/N), and tumor-liver signal intensity (T/L) ratios. RESULTS: T2-weighted TSE sequences provided better subjective image quality and reduced artifacts as compared with the T2-weighted CSE sequence. CSE and TSE sequences exhibited no statistically significant differences in liver S/N, lesion-liver C/N (CSE TE, 90 ms: 18.6 +/- 14.0; TSE TE, 90 ms: 16.5 +/- 12.9) and the detectability of focal liver lesions. Heavily T2-weighted TSE with a TE of 140 ms allowed correct characterization of focal liver lesions based on a T/L ratio of 3.0 in 84% of patients. CONCLUSIONS: T2-weighted TSE sequences are as suited as CSE for the detection (TE, 90 ms), and appear to be superior for the characterization (TE, 140 ms), of focal hepatic lesions. Whether a single sequence, such as a double-echo TSE or a single-echo TSE sequence with a TE between 110 and 120 ms, might perform both functions as well or better than CSE is unknown. However, because of time savings, TSE eventually may be preferred over CSE.

Artifacts↗
PubMed1996

[Comparison of fast turbo-spin-echo and gradient- and spin-echo sequences as well as echo planar imaging with conventional spin-echo sequences in MRI of focal liver lesions at 1.0 tesla].

PURPOSE: To evaluate the value of rapid T2-weighted turbo-spin sequences (TSE), turbo-spin-echo sequences with shortened echo spacing (UTSE), gradient-and-spin-echo sequences (GraSE) and T1-weighted echo-planar imaging (EPI) in comparison with conventional spin-echo sequences (SE) in the diagnosis of focal liver lesions. METHODS: 20 patients with malignant focal liver lesions underwent magnetic resonance imaging at 1.0 tesla. RESULTS: The use of fast T2-sequences (TSE, UTSE, GraSE) reduced the examination time to about 35-50%. Artifacts were reduced compared with the conventional T2-SE sequence. Quantitative analysis demonstrated that UTSE imaging had the highest tumor/liver contrast-to-noise ratio (CNR) followed by TSE, T2-SE and GraSE sequences. CNR with EPI was lower than with the T1-SE sequence (p < 0.05). CONCLUSION: Turbo-spin-echo sequences with shortened echo spacing yield a shorter imaging time and improved image quality without loss of signal intensity in tumor/liver-CNR, compared with conventional T2-pulse sequences in liver imaging at 1.OT.

Adult↗
PubMedDate not supplied

Prospective comparison of fast SE and GRASE sequences and echo planar imaging with conventional SE sequences in the detection of focal liver lesions at 1.0 T.

PURPOSE: Our goal was to investigate the value of T2-weighted fast SE (FSE) sequences, FSE sequences with shortened echo spacing (UFSE), combined gradient and spin echo sequences (GRASE), and segmented T1-weighted echo planar imaging (EPI) in comparison with conventional SE sequences in the detection of focal liver lesions. METHOD: Thirty-five patients with malignant focal liver lesions underwent MRI at 1.0 T. RESULTS: All fast T2-weighted imaging techniques (FSE, UFSE, GRASE) showed fewer artifacts and overall better image quality than the conventional SE sequence. Quantitative analysis demonstrated that UFSE imaging had the highest tumor/liver contrast/noise ratio (C/N) (13.9 +/- 5.5) followed by FSE (12.7 +/- 4.5), T2 SE (10.9 +/- 4.2), and GRASE (10.0 +/- 4.8) sequences. The differences in C/N between the UFSE and T2 SE sequences was statistically significant (p < 0.05). C/N was significantly higher (p < 0.01) for the T1 SE (-7.4 +/- 3.8) than for the EPI sequence (-0.5 +/- 5.6). CONCLUSION: FSE sequences with shortened echo spacing are valuable for liver imaging at 1.0 T. In comparison with the T2 SE sequence, they yielded better image quality and comparable tumor/liver C/N.

Adult↗
PubMed1988

Sequence-dependent variability of DNA structure. Influence of flanking sequences and fragment length on digestion by conformationally sensitive nucleases.

DNase I and 1,10-phenanthroline-copper are two nucleolytic activities which are sequence-dependent in their scission reaction yet are not nucleotide-specific at their site of cutting. When these two nucleases are used to digest identical sequences in 18-base pair oligonucleotides and in restriction fragments 10-fold longer, the digestion patterns are similar at sequence positions in the interior of the fragment. Changes in reactivity to 1,10-phenanthroline-copper associated with mutational changes in the lac promoter in biochemically functional restriction fragments are duplicated in 18-base pair oligonucleotides. The structural variability of a given DNA sequence detected by these conformationally sensitive nucleolytic activities is therefore encoded in local sequence and not sensitive to fragment length. Digestion patterns of a repeated 7-base pair sequence within a longer sequence have the same characteristic except for the two nucleotides at the 5' periphery of the direct repeat. This conclusion is based on the digestion pattern of a restriction fragment which contains the polyadenylation site of the mouse immunoglobulin mu heavy chain gene. Two pairs of different 7-base pair sequences repeated in this fragment retain their distinctive digestion patterns. DNA sequences which comprise the binding sites of regulatory proteins, retain a characteristic structure only influenced at their peripheries by two to three bases of the flanking sequence.

DNA↗
PubMed1991

A single-stranded DNA binding protein from S. cerevisiae specifically recognizes the T-rich strand of the core sequence of ARS elements and discriminates against mutant sequences.

A protein named ssARS-T binding protein has been purified from yeast that specifically binds to the T-rich strand of the consensus core sequence of yeast autonomously replicating sequence (ARS) elements. As assayed from gel mobility shift experiments the ssARS-T protein shows characteristics of a sequence specific single-stranded DNA binding protein. The complementary A-rich strand of the ARS core sequence is bound much more weakly and no binding can be detected for the double-stranded form of the core sequence. Three single base substitutions in the core sequence that are known to abolish ARS function in vivo also lead to weaker binding of the core sequence to the ssARS-T protein in vitro. The strong correlation between the binding of mutated sequences in vitro and the ARS properties of these sequences in vivo points to an essential function of the ssARS-T protein during replication initiation in yeast ARS elements.

Base Sequence↗
PubMed2005

ITS1 sequence variabilities correlate with 18S rDNA sequence types in the genus Acanthamoeba (Protozoa: Amoebozoa).

The subgenus classification of the ubiquitously spread and potentially pathogenic acanthamoebae still poses a great challenge. Fifteen 18S rDNA sequence types (T1-T15) have been established, but the vast majority of isolates fall into sequence type T4, and so far, there is no means to reliably differentiate within T4. In this study, the first internal transcribed spacer (ITS1), a more variable region than the 18S rRNA gene, was sequenced, and the sequences of 15 different Acanthamoeba isolates were compared to reveal if ITS1 sequence variability correlates with 18S rDNA sequence typing and if the ITS1 sequencing allows a differentiation within T4. It was shown that the variability in ITS1 is tenfold higher than in the 18S rDNA, and that ITS1 clusters correlate with the 18S rDNA clusters and thus corroborate the Acanthamoeba sequence type system. Moreover, high sequence dissimilarities and distinctive microsatellite patterns could enable a more detailed differentiation within T4.

Acanthamoeba↗