Evaluation of a new molecular assay for detection of human immunodeficiency virus type 1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA.

BACKGROUND: Rapid, sensitive, specific, and cost-effective screening of donated blood to prevent transmission of infectious agents remains challenging. In recent years, incorporation of nucleic acid testing for HIV-1 and HCV RNA improved blood safety by reducing the window period between infection and serologic detection. For HBV infection, this window period with most serologic assays is 50-60 days. Adding a nucleic acid test (NAT) for HBV DNA with existing NATs for HIV-1 and HCV RNA would further improve blood safety and blood screening efficiency. OBJECTIVE: To evaluate the Procleix Ultrio Assay for simultaneous detection of HIV-1 and HCV RNA and HBV DNA and corresponding discriminatory assays. STUDY DESIGN: The performance of these assays, which utilize the same technology and assay format as the Procleix HIV-1/HCV assay, was determined using relevant clinical specimens and analytical sensitivity and specificity panels. RESULTS: The Procleix Ultrio Assay demonstrated specificity of > or =99.5% in healthy donor blood specimens and in plasma containing potentially interfering substances or other blood-borne pathogens. Assay sensitivity demonstrated >95% detection of 100copies/mL, 30IU/mL, and 15IU/mL for HIV-1 and HCV RNA, and HBV DNA, respectively. The assay detects all known HIV-1 subtypes and HCV and HBV genotypes and is highly reproducible. Statistical analysis using receiver operating characteristic plots demonstrated wide analyte cutoff values for each assay associated with assay specificity and sensitivity of > or =99.5%. CONCLUSIONS: In this investigational study, the Procleix Ultrio Assay sensitivity and specificity were similar to existing NATs used in blood-bank settings to detect HIV-1 and HCV RNA and provided equivalent sensitivity and specificity for detection of HBV DNA. Using this combination assay, blood safety may be improved and the multiplex format enhances blood screening efficiency. The throughput capability of this assay is compatible with large volume processing and the chemistry is adaptable to full automation.

Mycorrhizal diversity in photosynthetic terrestrial orchids.

• Specific orchid-fungal associations are known for nonphotosynthetic orchids but fungal diversity in photosynthetic orchids is thought to be quite broad. Specific fungal associations will figure prominently in conservation efforts, while diverse associations may require less attention. We combined culture techniques with ITS and mtLSU sequences and phylogenetic analysis to determine the genetic diversity of mycorrhizal fungi associated with an evergreen, a spring-green, and a winter-green orchid and compared this diversity with that published for a nonphotosynthetic orchid. • Mycorrhizal diversity in two of the three photosynthetic orchids was lower than for the nonphotosynthetic orchid. Mycorrhizal diversity in protocorms of the third species was also equal to, or less than, the fungal diversity associated with the nonphotosynthetic species, but adult fungal diversity was greater. • We found that photosynthetic orchids do not necessarily have more diverse mycorrhizal associations than nonphotosynthetic orchids. Similarly, evergreen orchids do not necessarily have greater mycorrhizal diversity than seasonally green orchids. Thus, orchid mycorrhizal diversity may not be determined by adult photosynthetic capacity.

Validity of the hand test for diagnosing organicity in a clinical setting.

When validated against a clinical sample, the Hand Test was able to differentiate between 50 brain-damaged and 50 functionally disturbed individuals with 69% accuracy. Significant variables were Description, Withdrawal, Pathology, Automatic Phrasing, Gross, Hiding, and Demonstration. The Hand Test seems to have some value for diagnosing organicity, but it should be used with caution and preferably as part of a test battery.

Hybridization of common reed in North America? The answer is blowing in the wind.

BACKGROUND AND AIMS: We review evidence for hybridization of Phragmites australis in North America and the implications for the persistence of native P. australis ssp. americanus populations in North America. We also highlight the need for an updated classification system, which takes P. australis intraspecific variation and hybridization into account. METHODOLOGY: We reviewed available published, in press and in preparation literature to assess the likelihood of hybridization and interbreeding in genotypes of P. australis present in North America. PRINCIPAL RESULTS: Experimental results demonstrate that hybridization among introduced and native haplotypes is possible within the genus Phragmites, yet evidence that hybridization has occurred naturally is only starting to emerge. The lag in identifying hybridization in Phragmites in North America may be related to under-sampling in some parts of North America and to a lack of molecular tools that provide the capability to recognize hybrids. CONCLUSIONS: Our understanding of the gene flow within and between species in the genus Phragmites is moving at a fast pace, especially on the east and Gulf coasts of North America. More attention should also be focused on the Great Lakes region, the southwestern and the west coast of the USA, where sympatry has created opportunities for hybridization. Where hybridizations have been detected, there are currently no published data on how hybridization affects plant vigour, morphology, invasiveness or conservation of the genetic integrity of the North American native subspecies. We conclude that the detection of more hybridization is highly likely and that there is a need to develop new markers for the different Phragmites species and lineages to fill current knowledge gaps. Finally, we suggest that the classification system for P. australis should be updated and published to help clarify the nomenclature.

Low-frequency chimeric yeast artificial chromosome libraries from flow-sorted human chromosomes 16 and 21.

Construction of chromosome-specific yeast artificial chromosome (YAC) libraries from sorted chromosomes was undertaken (i) to eliminate drawbacks associated with first-generation total genomic YAC libraries, such as the high frequency of chimeric YACs, and (ii) to provide an alternative method for generating chromosome-specific YAC libraries in addition to isolating such collections from a total genomic library. Chromosome-specific YAC libraries highly enriched for human chromosomes 16 and 21 were constructed. By maximizing the percentage of fragments with two ligatable ends and performing yeast transformations with less than saturating amounts of DNA in the presence of carrier DNA, YAC libraries with a low percentage of chimeric clones were obtained. The smaller number of YAC clones in these chromosome-specific libraries reduces the effort involved in PCR-based screening and allows hybridization methods to be a manageable screening approach.

Polyamines eliminate an extreme size bias against transformation of large yeast artificial chromosome DNA.

The recent development of vectors and methods for cloning large linear DNA as yeast artificial chromosomes (YACs) has enormous potential in facilitating genome analysis, particularly because of the large cloning capacity of the YAC cloning system. However, the construction of comprehensive libraries with very large DNA segments (400-500 kb average insert size) has been technically very difficult to achieve. We have examined the possibility that this difficulty is due, at least in part, to preferential transformation of the smaller DNA molecules in the yeast transformation mixture. Our data indicate that the transformation efficiency of a 330-kb linear YAC DNA molecule is 40-fold lower, on a molar basis, than that of a 110-kb molecule. This extreme size bias in transformation efficiency is dramatically reduced (to less than 3-fold) by treating the DNA with millimolar concentrations of polyamines prior to and during transformation into yeast spheroplasts. This effect is accounted for by a stimulation in transformation efficiency of the 330-kb YAC molecule; the transformation efficiency of the 110-kb YAC molecule is not affected by the inclusion of polyamines. Application of this finding to the cloning of large exogenous DNA as artificial chromosomes in yeast will facilitate the construction of genomic libraries with significantly increased average insert sizes. In addition, the methods described allow efficient transfer of YACs to yeast strain backgrounds suitable for subsequent manipulations of the large insert DNA.

Danthonia Spicata (Poaceae) and Atkinsonella Hypoxylon (Balansiae): environmental dependence of a symbiosis.

Epiphytic and endophytic fungal infections often enhance plant growth. However, supporting active fungal tissue may be costly to plants in low-nutrient conditions and may affect the spatial distribution of host plants in heterogeneous environments. We examined the field distribution of Danthonia spicata infected and uninfected by the epiphytic fungus Atkinsonella hypoxylon relative to soil resource levels. We also conducted a greenhouse experiment to determine how D. spicata growth and performance responded to soil fertility and moisture. In two of three field populations, locations where A. hypoxylon occurred had higher ammonia, but lower soil moisture, than locations where D. spicata were uninfected. Infected and uninfected plants had similar growth rates across greenhouse treatments, but infected plants had a performance (size × survival) disadvantage relative to uninfected plants in high-nutrient, high-moisture and low-nutrient, low-moisture conditions. Field locations with D. spicata had low soil moisture, thus the performance disadvantage of infected plants in low-nutrient, low-moisture conditions corresponds to field observations that infected plants are rare in habitats with low ammonia. In a field common garden, infected plants had higher nitrogen concentrations than uninfected plants, suggesting that high nitrogen demand by A. hypoxylon may exclude infected plants from low-fertility field locations.

YAC cloning of DNA embedded in an agarose matrix.

Yeast artificial chromosome (YAC) cloning of DNA in agarose is an alternative method to cloning from aqueous solutions. It minimizes any shearing that may result from handling of high molecular weight DNA and can be done with nanogram to microgram amounts of material, which facilitates construction of YACs from sources of DNA other than genomic DNA isolated from cells. The average size of the YACs recovered (200-1000 kb) and efficiency of transformation of ligation products (200-1000 cfu/micrograms) are similar to those reported using aqueous protocols. This method has been used to construct chromosome specific YACs, and it should be possible to apply the technique to the construction of chromosome specific libraries using flow sorted chromosomes as source material, and the cloning of restriction fragments isolated by preparative pulsed field gel electrophoresis.

A method for coping with overall productivity when making between-group category comparisons.

A straightforward method for dealing with the effect of response productivity when comparing individual scoring categories between groups was presented along with an example. It was contended that percentage comparisons based on group data can circumvent the reliability problems associated with percentage scores derived from single protocols. Other problems connected with ratio and percentage scores were discussed.

Yeast artificial chromosome vectors for efficient clone manipulation and mapping.

The yeast artificial chromosome (YAC) cloning system allows the cloning of exogenous DNA several hundred kilobases in length. To enhance the usefulness of this technology, yeast artificial chromosome vectors have been designed for efficient clone characterization, manipulation, and mapping. The vectors contain a polylinker with unique EcoRI, BglII, NotI, EagI, SacII, SalI, NruI, NheI, and ClaI cloning sites and T7 bacteriophage promoters positioned to allow the generation of riboprobes from the exogenous DNA ends. Centric and acentric vector arms were constructed as separate plasmids to allow the recovery of both ends of the YAC insert DNA directly in Escherichia coli. In addition, YACs generated using this vector system contain a yeast gene (SUP 11) that allows visual monitoring of YAC stability and copy number.

Cloning and characterization of a novel human clathrin heavy chain gene (CLTCL).

An exon representing a novel clathrin heavy chain gene (CLTCL) was isolated during gene identification studies and transcription mapping of human chromosome 22. Isolation and sequencing of cDNA clones corresponding to this exon revealed extensive similarity of the predicted amino acid sequence of this gene product to those of clathrin heavy chain genes of other species. Northern blot analysis has revealed an apparent developmental expression pattern of an approximately 6-kb mRNA. The gene appears to be expressed ubiquitously in the limited number of fetal tissues that were tested, but is selectively expressed in certain adult tissues, particularly in skeletal muscle. In addition, alternative splicing of an exon was observed near the carboxyl terminus of the predicted gene product. Its location overlaps the domain putatively involved in clathrin light chain binding and is adjacent to the heavy chain self-assembly (or trimerization) region, suggesting that alternative splicing may be involved in regulating one or both of these interactions. The expression pattern of this gene, in addition to its potential role in receptor-mediated endocytosis and signal transduction, suggests that it may be important in some developmental processes. The location of CLTCL on human chromosome 22 near the region commonly deleted in DiGeorge and other apparent haploinsufficiency syndromes warrants further investigation into its relationship with these developmental disorders.

Characterization of msim, a murine homologue of the Drosophila sim transcription factor.

Mutations in the Drosophila single-minded (sim) gene result in loss of precursor cells that give rise to midline cells of the embryonic central nervous system. During the course of an exon-trapping strategy aimed at identifying transcripts that contribute to the etiology and pathophysiology of Down syndrome, we identified a human exon from the Down syndrome critical region showing significant homology to the Drosophila sim gene. Using a cross-hybridization approach, we have isolated a murine homolog of the Drosophila sim gene, which we designated msim. Nucleotide and predicted amino acid sequence analyses of msim cDNA clones indicate that this gene encodes a member of the basic-helix-loop-helix class of transcription factors. The murine and Drosophila proteins share 88% residues within the basic-helix-loop-helix domain, with an overall homology of 92%. In addition, the N-terminal domain of MSIM contains two PAS dimerization motifs also featured in the Drosophila sim gene product, as well as a small number of other transcription factors. Northern blot analysis of adult murine tissues revealed that the msim gene produces a single mRNA species of approximately 4 kb expressed in a small number of tissues, with the highest levels in the kidneys and lower levels present in skeletal muscle, lung, testis, brain, and heart. In situ hybridization experiments demonstrate that msim is also expressed in early fetal development in the central nervous system and in cartilage primordia. The characteristics of the msim gene are consistent with its putative function as a transcriptional regulator.

Construction and characterization of a YAC library with a low frequency of chimeric clones from flow-sorted human chromosome 9.

Human chromosome 9 DNA, flow-sorted from somatic cell hybrid PK-87-9, has been used to construct two complete digest YAC libraries. The combined representation of chromosome 9 in these libraries, estimated by hybridization of chromosome 9-specific sequences to YAC colony grids, is approximately 95%. The frequency of chimeric clones, analyzed by fluorescence in situ hybridization of chromosome 9 YACs to human metaphase chromosomes, was estimated to be approximately 4%. These libraries provide a resource for physical mapping and for moving from genetic markers to disease loci on chromosome 9.

Construction of human chromosome 21-specific yeast artificial chromosomes.

Chromosome 21-specific yeast artificial chromosomes (YACs) have been constructed by a method that performs all steps in agarose, allowing size selection by pulsed-field gel electrophoresis and the use of nanogram to microgram quantities of DNA. The DNA sources used were hybrid cell line WAV-17, containing chromosome 21 as the only human chromosome and flow-sorted chromosome 21. The transformation efficiency of ligation products was similar to that obtained in aqueous transformations and yielded YACs with sizes ranging from 100 kilobases (kb) to greater than 1 megabase when polyamines were included in the transformation procedure. Twenty-five YACs containing human DNA have been obtained from a mouse-human hybrid, ranging in size from 200 to greater than 1000 kb, with an average size of 410 kb. Ten of these YACs were localized to subregions of chromosome 21 by hybridization of RNA probes (corresponding to the YAC ends recovered in Escherichia coli) to a panel of somatic cell hybrid DNA. Twenty-one human YACs, ranging in size from 100 to 500 kb, with an average size of 150 kb, were obtained from approximately equal to 50 ng of flow-sorted chromosome 21 DNA. Three were localized to subregions of chromosome 21. YACs will aid the construction of a physical map of human chromosome 21 and the study of disorders associated with chromosome 21 such as Alzheimer disease and Down syndrome.

Clinical, cytogenetic, and molecular genetic characterization of two unrelated patients with different duplications of 21q.

We present 2 patients with dup(21q). Patient MP01 had mild mental retardation, facial findings characteristic of Down syndrome (DS), and a terminal duplication of chromosome 21. His karyotype was 46,XY,dup(21) (q22.1-qter). Patient MP03 had mild mental retardation, minor anomalies not characteristic of DS, and a duplication of the proximal long arm of chromosome 21, karyotype 46,XX,dup(21) (q11.2-q21.2). The patients were studied with single-copy DNA sequences from 20 loci on chromosome 21 to characterize the extent of the duplicated regions at the DNA level. DNA loci from D21S55 to COL6A1 were triplicated in patient MP01 while loci from D21S13 to D21S8 were triplicated in patient MP03. Our results support the hypothesis of a critical region of chromosome 21, which in triplicate is responsible for many of the facial changes associated with DS. Other genes outside this region may also contribute to other abnormalities observed in DS.

Clinical, cytogenetic, and molecular evaluation of a patient with partial trisomy 21 (21q11-q22) lacking the classical Down syndrome phenotype.

The clinical, cytogenetic, and molecular studies of an individual are presented here for the purpose of further characterizing what regions of chromosome 21q are essential for expression of the typical Down syndrome phenotype. This individual had a de novo, unbalanced translocation chromosome interpreted as: 45,XX,t(18;dup[21q]). Physical examination revealed mild manifestations, but not the typical phenotype of Down syndrome. The patient was studied using 20 single copy probes known to map to the 21q region. DNA polymorphism and dosage analyses showed triplication of loci D21S13 through D21S58 involving 21q11 to 21q22.1, and possibly involving the 21q22.2 region. Her clinical presentation, which did not include the classical findings of Down syndrome, suggests that regions distal to 21q22.1, when present in triplicate dose, account for the major manifestations of the classical phenotype. However, duplication of the area between the centromere and 21q22.1 may contribute to some of the other abnormalities in Down syndrome.

Mapping of low-frequency chimeric yeast artificial chromosome libraries from human chromosomes 16 and 21 by fluorescence in situ hybridization and quantitative image analysis.

Yeast artificial chromosome (YAC) clones from low-frequency chimeric libraries of human chromosomes 16 and 21 were mapped onto human diploid fibroblast metaphase chromosomes using fluorescence in situ hybridization (FISH) and digital imaging microscopy. YACs mapped onto chromosome 21 were selected to provide subregional location and ordering of known and unknown markers on the long arm of chromosome 21, particularly in the Down syndrome region (q22). YACs mapped onto chromosome 16 were selected to overlap regions spanning chromosome 16 cosmid maps. YAC clones were indirectly labeled with fluorescein, and the total DNA of the chromosome was counterstained with propidium iodide. A single image containing both the FISH signal and the whole chromosome was acquired for each chromosome of interest containing the fluorescent probe signal in a metaphase spread. From the digitized image, the fluorescence intensity profile through the long axis of the chromosome gave the total chromosome length and the probe position. The map position of the probe was expressed as the fractional length (FL) of the total chromosome relative to the end of the short arm (FLpter). From each clone hybridized, 20-40 chromosome images were analyzed. Thirty-eight YACs were mapped onto chromosome 16, and their FLs were distributed along the short and long arms. On chromosome 21, 47 YACs were mapped, including 12 containing known markers. To confirm the order of a dense population of YACs within the Down syndrome region, a two-color mapping strategy was used in which an anonymous YAC was located relative to one or two known markers on the metaphase chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of 102 exons to a 2.5 Mb region involved in Down syndrome.

Exon amplification has been applied to a 2.5 Mb region of chromosome 21 that has been associated with some features of Down syndrome (DS). Identification of the majority of genes from this region will facilitate the correlation of the over-expression of particular genes with specific phenotypes of DS. Over 100 gene fragments have been isolated from this 2.5 Mb segment. The exons have been characterized by sequence analysis, comparison with public databases and expansion to cDNA clones. Localization of the exons to chromosome 21 has been determined by hybridization to genomic Southern blots and to YAC and cosmid clones representing the region. This has resulted in a higher resolution physical map with a marker approximately every 25 kb. This integrated physical and transcript map will be valuable for fine mapping of DNA from individuals with partial aneuploidy of chromosome 21 as well as for assessing and ultimately generating a complete gene map of this segment of the genome.

The COL6A1 and COL6A2 genes exist as a gene cluster and detect highly informative DNA polymorphisms in the telomeric region of human chromosome 21q.

The genes that encode the alpha 1 (VI) and alpha 2 (VI) collagen chains, designated COL6A1 and COL6A2, map to human chromosomal band 21q22.3. Using pulsed-field gel electrophoresis and somatic cell hybrids, we found that COL6A1 and COL6A2 form a gene cluster on the most distal part of chromosome 21. Furthermore, we detected several DNA polymorphisms (both restriction site and VNTRs) associated with these loci. These polymorphisms make the COL6A1 and COL6A2 genes among the most informative markers on human chromosome 21.

Physical mapping of the 5 Mb D9S196-D9S180 interval harboring the basal cell nevus syndrome gene and localization of six genes in this region.

The basal cell nevus syndrome (Gorlin syndrome) is characterized by multiple basal cell carcinomas and diverse developmental defects. The gene responsible for this syndrome has been mapped previously to a 2 cM interval between D9S196 and D9S 180 at 9q22.3, and very recently mutations of a candidate gene in this region--the human homolog of the Drosophila patched gene have been identified. We report here on physical mapping studies integrating a contig of yeast artificial chromosomes and bacterial artificial chromosomes with a long-range map spanning approximately 5 Mb between the recombination-determined flanking markers. Six genes have been mapped to this interval.