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Biomedical subjects

F Grosveld

Publications and source records attributed to F Grosveld.

At least 181 records · Page 10Linked to original sources

Cloning and developmental expression of the murine neurofilament gene family.

DNA clones encoding the 3 mouse neurofilament (NF) genes have been isolated by cross-hybridization with a previously described NF-L cDNA probe from the rat. Screening of a lambda gt10 cDNA library prepared from mouse brain RNA led to the cloning of an NF-L cDNA of 2.0 kb that spans the entire coding region of 541 amino acids and of an NF-M cDNA that covers 219 amino acids from the internal alpha-helical region and the carboxy-terminal domains of the protein. These cDNA clones were used as probes to screen mouse genomic libraries, and cosmid clones containing both NF-L and NF-M sequences were isolated as well as overlapping cosmids containing the NF-H gene. This strongly suggests that the 3 neurofilament genes are organised in a cluster and derived by gene duplication of a common ancestral gene. RNA blot analyses using specific DNA probes for each of the genes indicate that NF mRNAs are differentially expressed during brain development. The NF-L and NF-M mRNAs are detected early in the embryonal brain, with a progressive increase in their levels during development, while the NF-H mRNA is barely detectable at embryonal stages and accumulates later in the postnatal brain.

Animals↗

Specific detection of neuronal cell bodies: in situ hybridization with a biotin-labeled neurofilament cDNA probe.

We have used a biotinylated, 300-nucleotide cDNA probe which encodes the 68,000 MW neurofilament protein to detect neurofilament-specific mRNA in situ. The neurofilament message specifically demonstrates the neuronal cell bodies, in contrast to the usual antibody staining which detects their neurites. The hybridization is detected only in neuronal structures. Consequently, detection of the biotinylated neurofilament DNA probe by silver-intensified streptavidin-gold can be specifically used to identify neuronal cell bodies.

Animals↗

Cloning of a cDNA encoding the smallest neurofilament protein from the rat.

We have cloned a cDNA coding for the smallest rat neurofilament protein. The cDNA is 861 nucleotides long coding for 287 amino acids from the internal alpha-helical region and the carboxy-terminal tail domain of the neurofilament protein. Comparison of the porcine, mouse and rat neurofilament protein sequences shows that the protein is highly conserved (greater than 93% identity). Blot analysis indicates that the cDNA is derived from a single neurofilament gene that codes for two different poly(A)+ mRNA species.

Amino Acid Sequence↗

Differential expression of human globin genes introduced in K562 cells.

We have constructed a series of hybrid globin genes between the 5' half of the human epsilon-, gamma- and beta-globin genes, and the 3' half of the rabbit beta-globin gene. These hybrid genes were introduced into the cell line K562. Analysis of the hybrid mRNA populations show that only the hybrid epsilon- and gamma-globin genes are expressed in these cells in concordance with the endogenous K562 globin gene expression. It is therefore likely that these cells contain transcriptional factors which allow the specific expression of introduced epsilon- and gamma-, but not beta-globin genes, irrespective of their chromosomal localization.

Animals↗

Structure of the murine Thy-1 gene.

We have cloned the murine Thy-1.1 (AKR) and Thy-1.2 (Balb/c) genes. The complete exon/intron structure and the nucleotide sequence of the Thy-1.2 gene was determined. The gene contains four exons and three intervening sequences. The complete transcriptional unit gives rise to a tissue and developmental stage-specific mRNA of 1850 bp. The 5' end of the gene has multiple initiation sites and a non-TATA box promoter. The 3' end shows a single polyadenylation site after a very long untranslated region.

Amino Acid Sequence↗

HLA-DR genotyping by restriction fragment length polymorphism analyses.

We have established unique restriction fragment length polymorphism (RFLP) patterns characteristic of homozygous typing cells (HTCs) for HLA-DR-1 through HLA-DR-8 haplotypes. These RFLP patterns were found to segregate in family members and correlate 100% with HLA-DR antibody phenotyping. The RFLP patterns were used to type chronic myelocytic leukemic cells which have a Philadelphia translocation from 23 randomly selected Caucasoid patients. The results show an alternative method for the determination of the HLA-DR types without using live cells and to study disease association with the HLA-DR region.

Cell Line↗

Chromosomal localization of the human Thy-1 gene.

We have isolated the gene coding for human Thy-1. Introduction of this gene into HeLa cells by DNA-mediated transfer results in the expression of Thy-1 antigen on the cell surface. Chromosomal mapping of the Thy-1 gene by hybridization to metaphase chromosomes and Southern blots of DNA from hybrid cells indicate that the Thy-1 gene is located on the long arm of chromosome 11.

Antigens, Surface↗

Anti-Thy-1 antibody responses evoked by Thy-1 antigen expressed in transfected mouse mastocytoma cells and rat fibroblast.

The mouse genomic Thy-1.1 gene was isolated from a phage library constructed from AKR/J (Thy-1.1) mouse DNA. Partial nucleotide sequence analysis of the coding region showed that it has only a single nucleotide difference from the Thy-1.2 gene, namely that amino acid 89 reads CGA (Arg) in Thy-1.1 and CAA (Glu) in Thy-1.2, corresponding to the amino acid substitutions previously identified. It was subcloned into an SV-40 derived vector for transfection. Transient transfection into HeLa cells gave 2% positive staining by immunofluorescence. The gene in this vector was also co-transfected into L cells and mastocytoma cells (both of Thy-1.2 strain origin) together with the Agpt gene. L-cell clones selected for transformation proved almost negative for Thy-1.1 expression, and any positive clones gradually lost Thy-1.1 antigen expression in culture. On the contrary, all clones of mastocytoma transformants gave a high level of expression after more than 3 months in culture. The mastocytoma transformants were used to study the immunogenicity of Thy-1.1 molecules expressed on transfected cells. They evoked clear anti-Thy-1.1 plaque-forming cell (PFC) responses both in vivo and in vitro. The mastocytoma transformants also proved able to induce a T-dependent anti-Thy-1.1 antibody response in a cell transfer experiment. The immunogenicity of Thy-1.2 molecules on rat fibroblasts was also studied after transfection with a Thy-1.2 gene cosmid. Although Thy-1.2 expression was very low, these transfectants elicited a clear anti-Thy-1.2 PFC response from AKR spleen cells hyperimmunized against CBA thymocytes.

Animals↗

DNA sequences required for regulated expression of beta-globin genes in murine erythroleukaemia cells.

We have introduced into murine erythroleukaemia (MEL) cells a series of human globin gene cosmids and two sets of hybrid genes constructed from the human beta-globin gene and the human gamma-globin or murine H-2Kbm1 genes. S1-nuclease analysis of the mRNA products from these genes before and after MEL cell differentiation showed that the human beta-globin gene, but not the human epsilon- or gamma-globin or H-2Kbm1 genes, is induced specifically. Hybrid genes containing human beta-globin DNA sequences from either 5' or 3' side of the translation initiation site were both inducible. Measurement of the relative rate of transcription showed this induction to be the result of transcriptional activation. We therefore suggest that DNA sequences which regulate beta-globin gene expression during MEL differentiation are located both 5' and 3' to the translation initiation site.

Animals↗

DNA sequences required for regulated expression of beta-globin genes in murine erythroleukemia cells.

We introduced into MEL cells rabbit beta-globin gene deletion mutants and two sets of hybrid genes constructed from the inducible human beta-globin gene and noninducible human gamma-globin gene or the murine H-2Kbm1 class I MHC gene. S1 nuclease analysis of gene transcripts before and after MEL differentiation showed that induction of the rabbit beta-globin gene did not require more than 58 bp of DNA 5' to the transcription initiation site. Hybrid genes were constructed with human beta-globin DNA sequences from either 5' or 3' of the translation initiation site linked to the complementary parts of the gamma or H2Kbm1 genes. Both types of constructs were inducible during MEL differentiation. The relative rates of transcription of the 5' gamma-3' beta and 5'H2-3' beta hybrid genes show that induction of the hybrid gene transcripts results at least in part from transcriptional activation of the genes. We suggest that DNA sequences that regulate beta-globin gene transcription during MEL differentiation are located both 5' and 3' to the translation initiation site.

Animals↗

Unexpected relationships between four large deletions in the human beta-globin gene cluster.

Two independent gamma delta beta-thalassemias are each associated with large deletions. We show, by comparing DNA sequences, that the deletions are due to non-homologous DNA exchanges. The 5' breakpoints are located approximately the same distance apart and in the same order along the DNA as their 3' breakpoints. Two independent cases of hereditary persistence of fetal hemoglobin, also involving large deletions, show the same unexpected relationship between their 5' and 3' breakpoints. This relationship is most simply explained if, within each pair, the deletions are of approximately the same length. The results suggest that the four deletions were generated by a common mechanism. Perhaps their 5' and 3' breakpoints are physically close in the nucleus, although far apart on the linear DNA.

Base Sequence↗

Sequences related to HLA-DR alpha chain on human chromosome 6: restriction enzyme polymorphism detected with DC alpha chain probes.

Three sets of cosmid clones--containing the HLA-DR alpha chain gene and two additional related genes--were isolated from human genomic DNA libraries by using a cDNA probe for the HLA-DR alpha chain. Southern blot analysis using DNA from somatic cell hybrids indicated that all of the clones mapped to chromosome 6. Partial sequence analysis showed that the two additional related genes were highly homologous to each other, and to the HLA-DR alpha chain, in parts of the exon that encoded the alpha 2 domain but were more divergent in intron sequences. One of the genes corresponds to the HLA-DR-related DC series. DNA probes made from this gene revealed marked restriction enzyme polymorphism when hybridized to genomic DNA from HLA-DR typed homozygous cell lines. The patterns obtained from a number of homozygous and heterozygous cell lines correlated with the HLA-DR crossreactive serotypes and also indicated that there is a further sequence in the haploid human genome that is closely homologous with the DC alpha chain sequence. One family was studied and showed the expected HLA-DR-associated inheritance of restriction enzyme patterns. No polymorphism has yet been demonstrated in restriction enzyme fragments that include the other cloned sequence, which may correspond to the SB alpha chain gene or to a novel HLA-DR-related gene. These experiments indicate that there are at least three sequences in the human genome related, but not identical, to the HLA-DR alpha chain gene.

Base Sequence↗

Isolation of human oncogene sequences (v-fes homolog) from a cosmid library.

To define the human homolog (or homologs) of transforming sequences (v-fes gene) common to Gardner (GA) and Snyder Theilen (ST) isolates of feline sarcoma virus (FeSV), a representative library of human lung carcinoma DNA in a cosmid vector system was constructed. Three cosmid clones were isolated containing GA/ST FeSV v-fes homologous cellular sequences, within 32- to 42-kilobase cellular inserts representing 56 kilobases of contiguous human cellular DNA. Sequences both homologous to, and colinear with, GA or ST FeSV v-fes are distributed discontinuously over a region of up to 9.5 kilobases and contain a minimum of three regions of nonhomology representing probable introns. A thymidine kinase selection system was used to show that, upon transfection to RAT-2 cells, the human c-fes sequence lacked detectable transforming activity.

Bacteriophage lambda↗