Biomedical subjects
E Lai
Publications and source records attributed to E Lai.
Bacterial artificial chromosome cloning and mapping of a 630-kb human extrachromosomal structure.
We have cloned and mapped a circular 630-kb human extrachromosomal structure (termed amplisome) using the bacterial artificial chromosome (BAC) cloning system. Twenty-one BACs were isolated from an amplisome-enriched library by colony hybridization. The insert sizes range from 25 to 143 kb, with an average size of 82 kb. The coverage of the amplisome in clones is approximately 2.7-fold. To construct a physical map of the amplisome, we used three different but complementary methods: hybridization, STS content mapping, and fingerprinting. In addition, we compared the advantages and the drawbacks of these techniques in mapping the amplisomal BACs. The 21 BACs were grouped into two contigs and the two small gaps (3.5 and 26.5 kb) were filled by screening of a human genomic BAC library. The organization of the amplisome revealed by the BAC-based physical map is consistent with the long-range restriction map reported previously. Our results demonstrate that a 630-kb region can be rapidly cloned and mapped into contigs by use of the BAC system. Because of the low frequency (<0.1%) of chimerism and rearrangement, these BAC clones are ready for DNA sequencing and functional analysis.
The oncogene qin codes for a transcriptional repressor.
The retroviral oncogene qin codes for a protein that belongs to the winged helix family of transcriptional regulators. The Qin protein is localized in the nucleus and binds to the same DNA consensus sequence as rat brain factor 1 (BF-1). Cellular Qin shows greater affinity to DNA than does viral Qin. Alone or fused to the DNA-binding domain of the yeast GAL4 protein, both Qin proteins act as transcriptional repressors. The major transcriptional repression domain maps to the region of amino acids 252-395 of viral Qin.
Murine chromosomal location of eight members of the hepatocyte nuclear factor 3/fork head winged helix family of transcription factors.
A 100-amino-acid DNA-binding motif, known as the winged helix, was first identified in the mammalian hepatocyte nuclear factor-3 (HNF-3) and Drosophila fork head family of transcription factors. Subsequently, more than 40 different genes that contain the winged helix motif have been identified. In the studies described here, we have determined the murine chromosomal location of eight members of this gene family, HFH-1, HFH-3, HFH-4, HFH-5, HFH-6, HFH-8, BF-1, and BF-2, by interspecific backcross analysis. These genes, designated HNF-3 fork head homolog 1 (Hfh1), Hfh3, Hfh4, Hfh5, Hfh6, Hfh8, Hfh9, and Hfh10, respectively, mapped to 6 different mouse autosomes and are thus well dispersed throughout the mouse genome. Based on this mapping information, we predict the chromosomal location of these genes in humans and discuss the potential of these genes as candidates for uncloned mouse mutations.
Use of secondary pulsed field gel electrophoresis in separation of large DNA.
It has been reported that secondary pulsed field gel (SPFG) electrophoresis can dramatically increase the speed of separation of large DNA molecules without a decrease in resolution (Zhang, T. Y., Smith, C. L., and Cantor, C. R. (1991) Nucleic Acids Res. 19, 1291-1296). However, our attempts to duplicate previous SPFG conditions were unsuccessful. We therefore sought to more precisely define the effects of secondary pulsing on the separation of large DNA and to determine the value of the technique in separating molecules up to 1100 kb. Here we report on two of the key SPFG parameters, namely the frequency and duration of the secondary pulse on the migration and resolution of DNA in SPFG. We found that the size range of separation is determined by the sum of the duration of the primary and secondary pulses. Under optimal conditions, a 25-70% increase in velocity can be achieved without loss in resolution.
Pulsed field separation of large supercoiled and open-circular DNAs and its application to bacterial artificial chromosome cloning.
We have studied the separation of large (80-300 kbp) supercoiled (SC) DNA in conventional agarose gel electrophoresis, field inversion gel electrophoresis (FIGE) and pulsed field gel electrophoresis (PFGE). DNA migration was measured under a variety of electrophoretic conditions including different switch times, temperatures, agarose concentrations, and voltage gradients. The migration of SC DNA was found to be inversely proportional to its molecular weight in the three electrophoresis systems tested. In conventional agarose electrophoresis, voltage gradient was found to be the determining parameter in the separation of SC DNA. Unlike large linear DNAs, the migration of SC DNA was found to be independent of switch time in PFGE and FIGE. Broad DNA bands were observed in prolonged FIGE runs. In addition, we have also studied the migration of open-circular (OC) DNA (80 and 100 kbp) in pulsed field gel electrophoresis. Eighty kbp OC DNA can migrate into agarose gels under certain pulsed field conditions whereas 100 kbp OC DNA was trapped at the wells. Based on electrophoretic conditions described in this report, we can determine the size of bacterial artificial chromosome (BAC) clones without restriction enzyme digestion and have enriched the percentage of larger size clones in BAC cloning.
Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres.
We generated mice with a null mutation of the forebrain-restricted transcription factor BF-1 to examine its function in brain development. Heterozygous animals have an apparently normal phenotype. Homozygous null BF-1 mutants die at birth and have a dramatic reduction in the size of the cerebral hemispheres. The development of the ventral telencephalon is more severely affected than that of the dorsal telencephalon. Telencephalic neuroepithelial cells are specified in the BF-1 mutant, but their proliferation is reduced. Dorsal telencephalic neuroepithelial cells also differentiate prematurely, leading to early depletion of the progenitor population. These results suggest that BF-1 controls the morphogenesis of the telencephalon by regulating the rate of neuroepithelial cell proliferation and the timing of neuronal differentiation.
Adjustment of reported prevalence of respiratory symptoms for non-response in a multi-centre health survey.
BACKGROUND: Estimation of non-response bias by modelling prevalence as a function of the number of mailings required to achieve a response, or of the cumulative response, has been advocated, but the models have not incorporated age and sex, differential response rates by age and sex, or season of response. METHODS: The effect on age-sex standardized prevalence of estimating non-response bias using a variety of models was investigated using data on nine symptom and medication questions from 13,007 subjects in the three English centres of the European Community Respiratory Health Survey. Comparison was made of goodness of fit and the prediction of responses in a 25% follow-up sample with the observed values. RESULTS: Despite low response rates in Cambridge and significant decreases in prevalence with additional mailings or increasing cumulative response in Norwich, there were only small effects on estimated age-sex standardized prevalences. No model was consistently better for any centre or question. CONCLUSIONS: The models are useful for exploring the sensitivity of estimated prevalence to non-response bias, but should be used with caution to adjust estimates. Ideally first mailings should be staggered over the whole year so that mailing and season are not confounded, and sufficient mailings or other contacts carried out for the whole sample to ensure a high response rate.
Participation of small intraportal stem cells in the restitutive response of the liver to periportal necrosis induced by allyl alcohol.
To determine the involvement of different hepatocyte populations in response to periportal injury, the restitutive response to allyl alcohol (AA) injury was examined. Adult female Sprague-Dawley rats were injected intraperitoneally (IP) with 0.62 mmol/kg AA, killed at 6, 9, 12, 33, 57, 81, and 153 hours after injection, and the livers were examined for injury and for restitutive proliferation by histology, autoradiography, and immunohistochemistry to detect alpha-fetoprotein (AFP), glutathione-s-transferase-p (GST-p), desmin, leukocyte common antigen, albumin, and monoclonal antibodies to liver cells: OV-6, H-4, and T-6. AA produces variable periportal liver necrosis predominantly at 6 to 12 hours. Proliferation of hepatocytes throughout the hepatic cord is seen early after injury in nonnecrotic areas: predominantly in zone II, but also in zones I and III, including some cells adjacent to the central vein. Within 2 to 3 days the necrotic zones are filled with small cells and by 1 week the liver architecture is essentially restored. During the active restitutive reaction from the immediate periportal rim the following cell phenotypes are seen: null cells: -->(AFP+, OV-6-, GST-p-) cells-->(AFP-, OV-6+, GST-p+) cells-->large (AFP-, OV-6-, GST-p-, H-4+) liver cells. Albumin staining was negative. We conclude that restitutive proliferation of periportal necrosis induced by AA appears to be accomplished by proliferation of intraportal (?stem) cells whose progeny differentiate and eventually repopulate the necrotic zone.
Rapid pulsed field separation of DNA molecules up to 250 kb.
Pulsed field gel electrophoresis (PFGE) is capable of resolving a wide size range of DNA molecules which would all co-migrate in conventional agarose gels. We describe pulsed field gel conditions which permit DNA fragments of up to 250 kilobases (kb) to be separated in only 3.5 h. The separations, which employ commercially available gel boxes, are achieved using conditions which deviate significantly from traditional pulsed field conditions. PFGE separations have been thought to require reorientation angles greater than 90 degrees to be effective. However, reorientation angles of 90 degrees and even less will resolve DNA fragments a few hundred kb and smaller approximately 5 x faster than with standard pulsed field conditions. The mobility of DNA fragments separated with 90 degrees reorientation angles is switch time-dependent, as is seen for DNA run with the commonly used reorientation angle of 120 degrees. With DNA fragments of several hundred kb and smaller, higher field strengths may be used, resulting in still greater increases in separation speed. The conditions described allow DNA from large insert bacterial clones, such as those using cosmid, Fosmid, P1, bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC) vectors, to be prepared, digested and analyzed on gels within a single working day.
Structure and organization of a stable extrachromosomal element in human cells.
We have determined the structure and organization of a 630-kb extrachromosomal element (amplisome) containing the dihydrofolate reductase-encoding gene (DHFR) in a methotrexate (MTX)-resistant human cell line, HeLa-Bu25-10B3. The size and copy number of amplisomes have previously been found to remain remarkably stable with or without selection. Both linear and open circular 630-kb amplisomes are present in these cells. We have been able to isolate the linear amplisomes after pulsed-field gel electrophoresis (PFGE), and transfect the amplisomes into MTX-sensitive recipient cells by electroporation, thus demonstrating that DNA as large as 630 kb can be transfected into mammalian cells. The NotI restriction site immediately upstream from DHFR on the circular amplisome is devoid of methylation, suggesting that it is transcriptionally active. Restriction mapping by PFGE reveals that there is only one copy of DHFR per amplisome and no repetitive structure is observed. The small size of the amplisomes, their stability and our ability to transfect large DNA molecules provide the necessary ingredients for the development of mammalian cloning vectors for large DNA fragments.
Peripheral facial nerve palsy after high-dose radioiodine therapy in patients with papillary thyroid carcinoma.
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Expression of winged helix genes, BF-1 and BF-2, define adjacent domains within the developing forebrain and retina.
One of the earliest events in the development of the central nervous system is the establishment of positional identity along the anteroposterior (A-P) axis of the neuroepithelium. In recent years, regulatory genes with regionally restricted expression in the neuroepithelium have been identified which are believed to specify its developmental fate. We have previously described Brain Factor-1 (BF-1), a winged helix (WH) transcription factor expressed in the telencephalic neuroepithelium (Tao and Lai, 1992) Neuron 8:957-966. Here we report the cloning of the mouse cDNA for a novel WH protein, BF-2. We show that BF-2 is a sequence-specific DNA binding protein with a binding specificity distinct from BF-1. Its expression in the CNS during embryogenesis is restricted to the rostral diencephalic neuroepithelium. The caudal boundary of BF-2 expression is at the zona limitans intrathalamica. Rostrally, the BF-2 expression domain is adjacent to that of BF-1. The expression domains of these two factors define a boundary within the developing forebrain neuroepithelium. The BF-1/BF-2 boundary also extends laterally to divide the optic stalk and the retina into nasal (anterior) and temporal (posterior) domains. These observations suggest that in addition to playing a role in the subdivision of the forebrain, these two WH factors may also function to establish positional information in the retinal neuroepithelium.
Organization of the V gene segments in mouse T-cell antigen receptor alpha/delta locus.
The mouse T-cell receptor (TCR) alpha/delta locus was mapped using 17 V alpha and 4 V beta subfamily-specific probes. Four complementary methods were used: (1) an estimate of the V gene repertoire by Southern blot analysis of genomic DNA with subfamily-specific probes; (2) an analysis of V gene segments deleted by TCR gene rearrangements from a panel of T-cell tumors and hybridomas; (3) an analysis of overlapping clusters of cosmid clones; and (4) an analysis of large DNA fragments separated by field-inversion gel electrophoresis. The alpha/delta locus spans about 1 Mb. The distance between the 3'-most V gene segment (V delta 1) and the delta constant gene (C delta) is no more than 150 kb. Sixty-six V gene segments have been mapped physically on cosmids. The members of individual V alpha gene segment subfamilies are dispersed throughout the locus. In contrast, the V delta gene segments V delta 1 to 5 are clustered at the 3' end of the V gene segment cluster. At least two DNA segment duplications, 45 to 80 kb in length, are present in the locus. These data provide information on the evolution of the alpha/delta locus and on organization features that might influence the expression of specific V gene segments in gamma delta cells.
Prevalence of asthma and asthma-like symptoms in young adults living in three east Anglian towns.
BACKGROUND: The European Community respiratory health survey is examining the prevalence of asthma and risk factors for asthma. AIM: As part of this multinational survey, a study was undertaken to determine the prevalence of asthma and asthma-like symptoms in young adults living in Cambridge, Ipswich and Norwich using a postal questionnaire. METHOD: A previously validated symptom questionnaire was sent to 2500 men and 2500 women aged 20-44 years living in and registered with a general practitioner in each of the three towns. RESULTS: In total, approximately 9000 adults responded. The prevalence of symptoms suggestive of asthma was found to be similar in the three towns. Of respondents, 8% reported having been woken by an attack of shortness of breath at some time in the last 12 months, higher than previously reported. Five per cent reported having had an asthma attack. CONCLUSION: General practitioners wishing to examine asthma prevalence in their own practice population could use a similar methodology.
Hepatocyte nuclear factor 3/fork head or "winged helix" proteins: a family of transcription factors of diverse biologic function.
A family of transcription factors, first identified as hepatocyte nuclear factors (HNF-3 alpha, -3 beta, and -3 gamma) and as a homeotic Drosophila mutant, fork head, has been intensively studied for the past 4 years. Important findings have emerged about the structure of the DNA-binding portion of the proteins as well as biologic discoveries about the diversity of the family and its implied role in early development.
Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5.
The three-dimensional structure of an HNF-3/fork head DNA-recognition motif complexed with DNA has been determined by X-ray crystallography at 2.5 A resolution. This alpha/beta protein binds B-DNA as a monomer, through interactions with the DNA backbone and through both direct and water-mediated major and minor groove base contacts, inducing a 13 degrees bend. The transcription factor fold is very similar to the structure of histone H5. In its amino-terminal half, three alpha-helices adopt a compact structure that presents the third helix to the major groove. The remainder of the protein includes a twisted, antiparallel beta-structure and random coil that interacts with the minor groove.
Identification of nine tissue-specific transcription factors of the hepatocyte nuclear factor 3/forkhead DNA-binding-domain family.
Hepatocyte nuclear factor (HNF)-3 alpha, -3 beta, and -3 gamma are liver transcription factors that mediate the coordinate expression of a number of hepatocyte-specific genes. The HNF-3 proteins share DNA-binding-domain homology among themselves and with the Drosophila homeotic protein forkhead (fkh). The HNF-3/fkh DNA-binding domain constitutes an uncharacterized protein motif that recognizes its cognate DNA binding site as a monomer. Additional HNF-3/fkh-related proteins are known to be required for determination events during embryogenesis in Drosophila and Xenopus. In this report, we describe the isolation of nine additional HNF-3/fkh homologue (HFH) clones from rodent tissue cDNAs by using both low-stringency hybridization and a polymerase chain reaction protocol. Many of the HFH genes exhibit a tissue-restricted expression pattern and are transcribed in tissues other than liver, including brain, kidney, lung, and intestine. The HNF-3/fkh motif therefore comprises a large gene family of transcription factors that play a role in tissue-specific gene regulation and development.