PubMed Health⌕ Search

Biomedical subjects

Y F Lau

Publications and source records attributed to Y F Lau.

68 records · Page 4Linked to original sources
PubMed1983

Versatile cosmid vectors for the isolation, expression, and rescue of gene sequences: studies with the human alpha-globin gene cluster.

We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers--SV2-gpt, SV2-DHFR, and SV2-neo--in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human alpha-globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of alpha-globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult alpha-globin genes were more actively expressed than the embryonic zeta-globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

Base Sequence↗
PubMed1983

Amplification of the human alpha-globin gene enhances its expression.

We investigated the effect of globin gene amplification on alpha-globin gene expression. Chinese hamster ovary cells were transformed with a plasmid containing the human alpha-globin gene linked to a functional dihydrofolate reductase gene, and the transformed cells were selected with increasing concentrations of methotrexate in the culture medium. A cell clone which was resistant to 0.1 mM methotrexate showed a 500-fold amplification of the transformed alpha-globin and DHFR genes. Both genes expressed high levels of their respective mRNAs. The mRNAs were functional and were translated to alpha-globin and DHFR proteins. Thus, one strategy for globin gene therapy is to achieve a high copy number of the transformed globin genes.

Animals↗
PubMed1981

Studies of mammalian chromosome replication. II. Evidence for the existence of defined chromosome replicating units.

Sister chromatids of metaphase chromosomes can be differentially stained if the cells have replicated their DNA semiconservatively for two cell cycles in a medium containing 5-bromodeoxyuridine (BrdU). When prematurely condensed chromosomes (PCC) are induced in cells during the second S phase after BrdU is added to the medium, the replicated chromosome segments show sister chromatid differential (SCD) staining. Employing this PCC-SCD system on synchronous and asynchronous Chinese hamster ovary (CHO) cells, we have demonstrated that the replication patterns of the CHO cells can be categorized into G1/S, early, early-mid, mid-late, and late S phase patterns according to the amount of replicated chromosomes. During the first 4 h of the S phase, the replication patterns show SCD staining in chains of small chromosome segments. The amount of replicated chromosomes increase during the mid-late and late S categories (last 4 h). Significantly, small SCD segments are also present during these later intervals of the S phase. Measurements of these replicated segments indicate the presence of characteristic chromosome fragment sizes between 0.2 to 1.2 micrometers in all S phase cells except those at G1/S which contain no SCD fragments. These small segments are operationally defined as chromosome replicating units of chromosomal replicons. They are interpreted to be composed of clusters of molecular DNA replicons. The larger SCD segments in the late S cells may arise by the joining of adjacent chromosomal replicons. Further application of this PCC-SCD method to study the chromosome replication process of two other rodents, Peromyscus eremicus and Microtus agrestis, with peculiar chromosomal locations of heterochromatin has demonstrated an ordered sequence of chromosome replication. The euchromatin and heterochromatin of the two species undergo two separate sequences of decondensation, replication, and condensation during the early-mid and mid-late intervals respectively of the S phase. Similar-sized chromosomal replicons are present in both types of chromatin. These data suggest that mammalian chromosomes are replicated in groups of replicating units, or chromosomal replicons, along their lengths. The organization and structure of these chromosomal replicons with respect to those of the interphase nucleus and metaphase chromosomes are discussed.

Animals↗
PubMed1980

Nucleolar activity in differentiated cells after stimulation.

Initiation of nucleolar organizer region (NOR) activity was observed by using the silver staining method at various times after activation or stimulation of differentiated cells. Two methods were used: (1) activation of human lymphocytes by treatment with phytohemagglutinin (PHA), and (2) cell-cell fusion of chick erythrocytes with squirrel monkey cells. An increase in NOR activity in lymphocytes was seen as early as 4 h after PHA treatment and between 10 and 22 h in the chick erythrocytes after fusion. In both systems, as the size of the dormant cell nucleus increased, the amount of silver staining increased until the silver-stained area approached that of cycling cells.

Animals↗
PubMed1980

Studies of mammalian chromosome replication. I. BrdU-induced differential staining patterns in interphase and metaphase chromosomes.

The patterns of differential staining based on the effects of BrdU-substitution in chromosomal DNA have been examined in both metaphase chromosomes and prematurely condensed chromosomes (PCC) of interphase Chinese hamster cells. Results indicate that differential staining may be obtained in chromosomes from all stages of the cell cycle and correspond to the semi-conservation mode of DNA replication. Such fidelity of differential staining in both interphase and metaphase chromosomes suggests that components essential for induction of differential staining are present throughout the cell cycle and chromosomes may contain similar structures and organization throughout the cycle.

Animals↗
PubMed1979

Observations on the synaptonemal complex in Armenian hamster spermatocytes by light microscopy.

Using the silver staining technique, in somatic and meiotic chromosomes of the Armenian hamster (Cricetulus migratorius), it is possible to stain synaptonemal complexes (SCs) and the nucleolus organizer regions (NORs) in early spermatocytes. There are five pairs of autosomes (Nos. 2, 4, 6, 7, and 8) which have terminally located NORs. Synaptonemal complexes and accessory structures present in the sex chromosomes within the sex vesicle can be easily observed using light microscopy.

Animals↗
PubMed1979

Cytological analyses of 14p+ variant by means of N-banding and combinations of silver staining and chromosome bandings.

An inherited human karyological variant (14p+) has been studied with a number of cytochemical techniques. The short arm of this variant chromosome 14 is nearly as long as the long arm, giving the chromosome a submetacentric to metacentric appearance. In conventionally Giemsa-stained preparations, maximum of three secondary constrictions can be observed the marker arm. The secondary constrictions are silver-positive in Ag-NOR preparations. However, the entire arm stains deeply in N-banded preparations. The 14p+ arm is also Q-negative, C-negative, G-negative, and R-positive with an almost homogeneous texture. The difference between N-banding and silver staining is interpreted as the result of gene activities of the ribosomal cistrons.

Azure Stains↗
PubMed1978

Combination of silver and fluorescent staining for metaphase chromosomes.

A convenient and reliable method for simulatneous visualization of silver staining (Ag-NOR) of the nucleolus organizers and fluorescent bandings in metaphase chromosomes is described. Studies employing this combined procedure on human chromosomes revealed that the Ag-NOR patterns may be characteristic for each chromosome of each individual.

Chromosomes↗
PubMed1977

Studies of the squirel monkey, Saimiri sciureus, genome. II. C-band characterization and DNA replication patterns.

Cytological analysis of a squirrel monkey indicates a considerable degree of chromosomal polymorphism. DNA synthetic patterns reveal late synthesis in chromosomal segments that are C-band negative and asynchrony in DNA replication between homologs. Location of a satellite DNA with a CsCl neutral buoyant density of 4.691 g/ml was mainly in the noncentromeric constitutive heterochromatin.

Animals↗
PubMed1976

Sister chromatid differential staining pattern in prematurely condensed chromosomes.

Application of sister chromatid differential (SCD) procedure on G1, S and G2 prematurely condensed chromosomes (PCC) of cells in the second and third cycle of DNA replication in medium containing BrdU reveals differential staining patterns characteristic of their respective stages in the cell cycle. These findings also suggest a structural similarity between PCC and metaphase chromosomes.

Animals↗
PubMed1976

Studies of the squirrel monkey, Saimiri sciureus, genome. I. Cytological characterization of chromosomal heterozygosity.

Chromosomal banding analyses have revealed two nonhomologous pericentric inversions in the telocentric group of chromosomes of a squirrel monkey, Saimiri sciureus. This individual is probably an intersubspecific hybrid whose parents originated from different geographical locations. The C-banding pattern shows polymorphism in location and amount of constitutive heterochromatin in at least four pairs of chromosomes. Ammoniacal silver (Ag-AS) staining specific for locating the nucleolar organizers indicates that these are located on only one pair of chromosomes and that there is a difference in the size of the nucleolus organizer regions between the homologs of this pair.

Animals↗