Biomedical subjects

G M Huang

Publications and source records attributed to G M Huang.

5 recordsLinked to original sources

PubMed1994

A high-throughput plasmid DNA preparation method.

Genome projects require a high-throughput method for DNA template preparation, which traditional protocols can rarely provide. We introduce here a new protocol for plasmid and cosmid DNA preparation based on alkaline denaturation in a 96-well format. The essential improvement made in this protocol on previous 96-well preparation includes the purification by Pro-Cipitate in 96-well microfilters. The yield and quality of DNA are sufficient for restriction digestion, radioactive sequencing, and automated fluorescent sequencing. It is also significantly more cost-effective than several of the commercial mini-prep kits.

DNA↗

PubMed1994

Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure.

Retrovirus-mediated gene transfer of the human beta-globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta-globin gene disorders. However, expression of the transduced beta-globin gene linked to its proximal cis-acting sequences (-0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the beta-locus control region (beta-LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta-like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta-globin gene, but initial attempts to incorporate beta-LCR derivatives in retroviral vectors resulted in the production of low-titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced beta-globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow-repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced beta-LCR derivatives in dimethylsulfoxide-induced murine erythroleukemia cells and to achieve ratios of human beta-globin/murine beta maj-globin mRNA, on a per gene basis, as high as 80%.

Animals↗

PubMed1994

Saccharomyces cerevisiae U14 small nuclear RNA has little secondary structure and appears to be produced by post-transcriptional processing.

Yeast U14 small nuclear (sn) RNA is required for normal processing of rRNA. The sequence and folding properties of U14 were analyzed in the present study, with the aim of defining the structures of natural U14 subspecies and characterizing the folding properties of free U14 RNA. Natural U14 was determined to consist of four subspecies of 125-128 nucleotides, none containing a 5'-cap structure. Length heterogeneity occurs at both ends and is presumed to reflect post-transcriptional processing of U14 precursors. Results from nuclease and chemical probing revealed that U14 has surprisingly little secondary structure overall. Three essential sequence elements conserved among all U14 RNAs occur in regions that are largely single-stranded, i.e. box C, box D, and a 13-nucleotide segment complementary to 18 S rRNA; a non-essential 14-nucleotide sequence complementary to 18 S rRNA is also unpaired. Two non-conserved segments required for activity are part of a stably folded 32-base domain that is unique to yeast U14. Finally, a 5'-, 3'-stem shown earlier to be required for U14 accumulation appears to exist only in precursors to U14 and not in protein-free mature RNA. The implications of these results are discussed in terms of U14 synthesis and function.

Base Sequence↗

PubMed1992

Accumulation of U14 small nuclear RNA in Saccharomyces cerevisiae requires box C, box D, and a 5', 3' terminal stem.

U14 is one of several nucleolar small nuclear RNAs required for normal processing of rRNA. Functional mapping of U14 from Saccharomyces cerevisiae has yielded a number of mutants defective in U14 accumulation or function. In this study, we have further defined three structural elements required for U14 accumulation. The essential elements include the U14-conserved box C and box D sequences and a 5', 3' terminal stem. The box elements are coconserved among several nucleolar small nuclear RNAs and have been implicated in binding of the protein fibrillarin. New mutational results show that the first GA bases of the box C sequence UGAUGA are essential, and two vital bases in box D have also been identified. An intragenic suppressor of a lethal box C mutant has been isolated and shown to contain a new box C-like PyGAUG sequence two bases upstream of normal box C. The importance of the terminal stem was confirmed from new compensatory base changes and the finding that accumulation defects in the box elements can be complemented by extending the terminal stem. The results suggest that the observed defects in accumulation reflect U14 instability and that protein binding to one or more of these elements is required for metabolic stability.

Base Composition↗

PubMed1990

Sacroepidural analgesia for post-operative pain relief in children.

For evaluation of the practicality of epidural analgesia for alleviation of post-operative pain, 66 class I or II patients with age ranging from 6 months to 12 years undergoing elective surgical procedures below the lower abdomen were enrolled for study during the period from January to April 1989. Before termination of general anesthesia a single dose of 0.25% bupivacaine, respectively at 0.25 mL/kg (Ideal body weight), 0.5 mL/kg, 0.75 mL/kg, 1.0 mL/kg and 1.25 mL/kg was given sacroepidurally to 5 groups of patients. Our results showed that for surgery below the lower abdomen a dose at 1.0 mL/kg was sufficient to suffice the need for relief of post-operative pain. At this dosage it achieved a neural block up to T8-T6 and provided with an analgesia that could last 6.0 +/- 2.1 hours. During the entire course there were no untoward effects such as hypotension, bradycardia, vomiting and shivering to come about. Therefore, sacroepidural analgesia with 0.25% bupivacaine at fitting single dose is safe and feasible in children as far as relief of post-operative pain for procedures below the lower abdomen is concerned.

Analgesia, Epidural↗