Biomedical subjects

D J Fu

Publications and source records attributed to D J Fu.

13 recordsLinked to original sources

PubMed2001

Genome of the bacterium Streptococcus pneumoniae strain R6.

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

Bacterial Proteins↗

PubMed1999

Chip-based genotyping by mass spectrometry.

Silicon chips with immobilized target DNAs were used for accurate genotyping by mass spectrometry. Genomic DNAs were amplified with PCR, and the amplified products were covalently attached to chip wells via N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB) chemistry. Primer annealing, extension, and termination were performed on a 1-microl scale directly in the chip wells in parallel. Diagnostic products thus generated were detected in situ by using matrix-assisted laser desorption ionization mass spectrometry. This miniaturized method has the potential for accurate, high-throughput, low-cost identification of genetic variations.

Base Sequence↗

PubMed1998

Sequencing exons 5 to 8 of the p53 gene by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption ionization time of flight mass spectrometry was used to sequence exons 5 to 8 of the human p53 gene. A single tube procedure was established for target amplification and mass spectrometric (MS) sequencing. The MS sequencing scheme is designed for high throughput and parallel sample processing, and is amenable to full automation. Reliable sequencing data were obtained using fmol sample amounts. The high resolution and accuracy of MS sequencing was demonstrated by direct sequencing of a heterozygous template.

Base Sequence↗

PubMed1997

Sequencing double-stranded DNA by strand displacement.

Duplex probes with five base single-stranded overhangs can capture dsDNA targets from type IIS restriction nuclease digests. Ligation generates a predesigned nick site, where DNA polymerase can generate sequencing ladders by strand displacement or nick translation in the presence of trace amounts of dideoxynucleotides. This allows dsDNA targets to be captured from mixtures and directly sequenced without subcloning, purification or denaturation.

Base Sequence↗

PubMed1996

A strategy for rapid and efficient DNA sequencing by mass spectrometry.

Two methods of solid-phase Sanger DNA sequencing followed by detection with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry are demonstrated. In one method, sequencing ladders generated on an immobilized synthetic template were resolved up to the 63-mer including the primer. Detection sensitivity and resolution were sufficient for sequence analysis in the given range. This approach is particularly suitable for comparative (diagnostic) DNA sequencing. A second method that has the potential for high throughput de novo DNA sequencing is also presented; it uses immobilized duplex probes with five-base single-stranded overhangs to capture an unknown DNA template serving as primers for Sanger DNA sequencing. The power of mass spectrometry is demonstrated not only by its very high speed, but also by its ability to identify sequences that are not readable using gel electrophoresis.

Base Sequence↗

PubMed1996

Efficient preparation of short DNA sequence ladders potentially suitable for MALDI-TOF DNA sequencing.

Duplex probes with five-base single-stranded overhangs were developed for positional sequencing by hybridization [Broude et al., Proc Natl Acad Sci USA 91:3072-3076, 1994]. The partially duplex probes can be employed to capture single-stranded oligonucleotide targets and form primer-template complexes. Recently we showed that partially duplex probes can prime Sanger sequencing reactions on immobilized, but non-ligated long single-stranded targets (approximately 500 nucleotide) [Fu et al., Proc Natl Acad Sci, in press]. Here immobilized, non-ligated partially duplex probes were used to capture and sequence short single-stranded targets. This strategy is capable of rapidly preparing large numbers of samples for future mass spectrometric DNA sequencing.

Base Sequence↗

PubMed1995

A DNA sequencing strategy that requires only five bases of known terminal sequence for priming.

We have previously reported an enhanced version of sequencing by hybridization (SBH), termed positional SBH (PSBH). PSBH uses partially duplex probes containing single-stranded 3' overhangs, instead of simple single-stranded probes. Stacking interactions between the duplex probe and a single-stranded target allow us to reduce the probe sizes required to 5-base single-stranded overhangs. Here we demonstrate the use of PSBH to capture relatively long single-stranded DNA targets and perform standard solid-state Sanger sequencing on these primer-template complexes without ligation. Our results indicate that only 5 bases of known terminal sequence are required for priming. In addition, the partially duplex probes have the ability to capture their specific target from a mixture of five single-stranded targets with different 3'-terminal sequences. This indicates the potential utility of the PSBH approach to sequence mixtures of DNA targets without prior purification.

Base Sequence↗

PubMed1994

Activity of the hammerhead ribozyme upon inversion of the stereocenters for the guanosine 2'-hydroxyls.

Two guanosine 2'-hydroxyls in the hammerhead RNA complex at positions G5 and G8 are critical for efficient cleavage by this RNA catalyst. These two functional groups are likely involved in the binding of the metal cofactor, or they are involved in specific interresidue hydrogen-bonding interactions. The importance of the stereochemical positioning of both critical 2'-hydroxyls was investigated by comparing the cleavage rates of three arabinosylguanine-substituted complexes (in which the positions of specific guanosine 2'-hydroxyls were stereochemically altered by inverting the C2' stereocenter) with that of the native complex, as well as with the rates of the dG- and dFG-substituted complexes [in which the 2'-hydroxyls are absent as the result of substitution by 2'-deoxyguanosine (dG) or 2'-deoxy-2'-fluoroguanosine (dFG)]. The G5araG and G8araG complexes exhibit dramatically different cleavage rates. The G5araG complex is essentially inactive, at least 10(5)-fold slower than the native complex. RNA cleavage by this analogue ribozyme is also 1000-fold slower than cleavage by either the G5dG or the G5dFG ribozyme, both of which lack the 2'-hydroxyl at G5. By comparison, catlytic efficiency of the G8araG complex as expressed by kcat/Km is comparable with that of the native complex and some 2 orders of magnitude more active than either the G8dG or the G8dFG complex.

Arabinose↗

PubMed1993

Importance of specific guanosine N7-nitrogens and purine amino groups for efficient cleavage by a hammerhead ribozyme.

Seven modified hammerhead ribozyme/substrate complexes have been prepared in which individual purine nitrogens, the guanine N7-, the guanine N2-, or the adenine N6-nitrogen, have been excised. The modified complexes were chemically synthesized with the substitution of a single 7-deazaguanosine (c7G), inosine (I), or nebularine (purine riboside, P) base analogue as appropriate for residues G5, G8, G12, A13, A14, or A15. Two of the base analogues, c7G5 and C7G8, occur in a 19-mer ribozyme, while the remaining three residues are present in a 24-mer substrate. Under stoichiometric conditions, four of the complexes, G5c7G, G8c7G, G12c7G, and A14P, are cleaved with relatively little change in rate when compared with the native complex. Two complexes, A13P and A15P, are cleaved some 6-8-fold slower than the native complex, while the G12I complex is reduced in rate by 50-fold. Steady-state kinetic analyses indicate that the cleavage efficiencies, as measured by kcat/KM values, for the G5c7G, G8c7G, and G12c7G complexes are only marginally reduced relative to the native complex. The values for the A13P, A14P, and A15P complexes are reduced by 25-, 15-, and 60-fold, respectively. These reductions in cleavage efficiency are primarily a result of lower kcat values. By comparison, the kcat/KM value for the G12I complex is decreased 450-fold relative to the native complex and is characterized by an 8-fold increase in KM and a kcat value that is reduced nearly 60-fold. These results indicate that the N2-amino group of G12 in the hammerhead ribozyme/substrate complex is critical for efficient cleavage activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗

PubMed1992

Importance of specific adenosine N7-nitrogens for efficient cleavage by a hammerhead ribozyme. A model for magnesium binding.

Five modified hammerhead ribozyme/substrate complexes have been prepared in which individual adenosine N7-nitrogens have been excised. The modified complexes were chemically synthesized with the substitution of a single 7-deazaadenosine (c7A) base analogue for residues A11, A14, A26, A27, or A28. Two of the base analogues, c7A11 and c7A14, occur in a 19-mer ribozyme, while the remaining three residues, c7A26, c7A27, and c7A28, are present in a 24-mer substrate. Under stoichiometric conditions, four of the complexes are cleaved with relatively little change in rate when compared with that of the native complex. However, the relative rate for the c7A11 complex is some 35-fold slower than that of the native complex. Steady-state kinetic analyses indicate that the cleavage efficiencies, as measured by kcat/KM, for the c7A14, c7A26, c7A27, and c7A28 complexes are reduced 18-fold, 10-fold, 34-fold, and 16-fold, respectively. These reductions in cleavage efficiency are primarily a result of lower kcat values. By comparison, the cleavage efficiency of the c7A11 complex is reduced more than 200-fold relative to that of the native complex, again primarily as a result of a lower kcat value. The results suggest that the N7-nitrogen of A11 in the hammerhead ribozyme/substrate complex is critical for efficient cleavage activity. The results of the present work, in combination with those from previous reports, indicate that five critical functional groups are located within the tetrameric sequence G10A11U12G13. A preliminary model for the binding of a single magnesium cofactor to this portion of the sequence is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1992

Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme.

Eight modified ribozymes of 19 residues have been prepared with individual purine amino or hydroxyl groups excised. The modified ribozymes were chemically synthesized with the substitution of a single 2'-deoxyadenosine, 2'-deoxyguanosine, inosine, or purine riboside for residues G10, A11, G13, or A14. Five of the modified ribozymes cleaved the 24-mer substrate with little change in rate as monitored by simple first-order kinetics. However, deletion of the 2-amino group at G10 (replacement with inosine) or deletion of either of the 2'-hydroxyls at G10 or G13 (replacement with 2'-deoxyguanosine) resulted in ribozymes with a drastic decrease in cleavage efficiency. Increasing the concentration of the Mg2+ cofactor from 10 mM to 50 mM significantly enhanced cleavage efficiency by these three derivatives. Steady-state kinetic assays for these three ribozymes indicated that the modifications result in both an increase in Km and a decrease in kcat. These results suggest that the exocyclic amino group at-G10 and the hydroxyls at G10 and G13 are important both for ribozyme-substrate binding and for the Mg(2+)-catalyzed cleavage reaction.

Base Sequence↗

PubMed1991

Lead poisoning in Hawaii: 1990.

Although lead (Pb) is one of the oldest known and most thoroughly described toxins, it continues to be a significant health hazard in 1990. There has been much progress in defining the nature and extent of low-level lead toxicity during the past decade. There continues to be insidious sources of lead toxicity in our environment, in water, food, paint and contaminated soil. As the epidemiology of lead poisoning is more clearly defined, toxicities are recognized as the result of lower and lower levels of exposure. Recognition of low-level lead exposure and the primary prevention of its effects on health requires a keen awareness of high-risk environments as well as the subtle symptoms and signs of lead poisoning. A high index of suspicion by primary care physicians plus government support are necessary to implement successful prevention programs.

Child, Preschool↗

PubMed1990

Lead poisoning: cause for alarm.

Lead (Pb) is everywhere in our environment and all children are exposed to lead to some degree. The Center for Disease Control (CDC) has recently declared that a blood lead level of 25 micrograms/dL and an erythrocyte protophoryrin of 35 micrograms/dL or above indicate an excessive absorption of lead in children and constitutes grounds for intervention. A study reported that about 780,000 (4%) of American children age 6 months to 5 years had blood lead levels of 30 micrograms/dL or higher. If the current level of 25 micrograms/dL had been used as a criterion in this study, the number of children involved would be much higher. It has been clearly stated by the American Academy of Pediatrics that ideally, all pre-school children should be screened for lead absorption by means of the erythrocyte protophoryrin test. The following is a review of lead poisoning in children and the serious consequences which may follow if it is not recognized early.

Centers for Disease Control and Prevention, U.S.↗