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Biomedical subjects

A F Scott

Publications and source records attributed to A F Scott.

13 recordsLinked to original sources
PubMed1991

Isolation of an active human transposable element.

Two de novo insertions of truncated L1 elements into the factor VIII gene on the X chromosome have been identified that produced hemophilia A. A full-length L1 element that is the likely progenitor of one of these insertions was isolated by its sequence identity to the factor VIII insertion. This L1 element contains two open-reading frames and is one of at least four alleles of a locus on chromosome 22 that has been occupied by an L1 element for at least 6 million years.

Alleles↗
PubMed1991

Reverse transcriptase encoded by a human transposable element.

L1 elements are highly repeated mammalian DNA sequences whose structure suggests dispersal by retrotransposition. A consensus L1 element encodes a protein with sequence similarity to known reverse transcriptases. The second open reading frame from the human L1 element L1.2A was expressed as a fusion protein targeted to Ty1 virus-like particles in Saccharomyces cerevisiae and shown to have reverse transcriptase activity. This activity was eliminated by a missense mutation in the highly conserved amino acid motif Y/F-X-D-D. Thus, L1 represents a potential source of the reverse transcriptase activity necessary for dispersion of the many classes of mammalian retroelements.

Base Sequence↗
PubMed1990

Allele specific oligonucleotide probes for mouse I-A region and gene: I-A beta.

MHC Class II gene expression is being intensively studied in vitro. In vivo class II gene expression that occurs during G.V.H.R. and allograft reaction has not been studied as well as the gene expression in vitro because such studies require unambiguous detection of graft or host specific MHC class II genes. Although this is possible by using allele specific oligonucleotide probes for individual class II genes, such probes for class II genes in mice have not been described before. We compared the nucleotide sequences of I-A beta genes from various haplotypes and selected a region of minimal homology in the 2nd exon of this gene. We synthesized two oligonucleotide probes corresponding to a 20 nucleotide stretch in the hypervariable region of the I-A beta 1 genes of H-2k and H-2d haplotype mice. These probes specifically detect I-A beta mRNA from mice of appropriate haplotypes. These allele-specific probes should help study the in vivo expression of graft or host specific I-A genes in G.V.H.R. or allograft reaction.

Animals↗
PubMed1990

The role of cell adhesion and migration in the in vitro invasiveness of mouse adrenal carcinoma cells.

Using the Matrigel invasion assay, we have examined the role of cell adhesion and migration in the invasiveness of two cell lines, DM and HSR, derived from the Y1 mouse adrenocortical tumor. The DM cells were metastatic and more invasive (10-fold) than the nonmetastatic HSR cells. The difference in invasiveness could not be ascribed to different levels of secreted type IV collagenolytic activity since HSR cells secreted higher levels of activity. Cells from the metastatic DM line showed greater motility to both laminin and fibronectin when compared to the HSR line. Furthermore, both Matrigel and laminin promoted the attachment and spreading of DM cells but they had little effect on the adhesion of the HSR cells. Electron microscopic examination revealed an increased ruffling of the cell membrane in the metastatic DM line. These studies suggest a role for cell adhesion and migration on the invasion of Matrigel by malignant tumor cells.

Adrenal Gland Neoplasms↗
PubMed1989

Characterization of a nondeleterious L1 insertion in an intron of the human factor VIII gene and further evidence of open reading frames in functional L1 elements.

We have characterized an insertional event in IVS-10 of the factor VIII gene in a pedigree containing a hemophilia A patient (JH-25). The inserted DNA is a 5' truncated L1 element that is 681 bp long followed by a 3'66-bp poly(A) tract. The L1 element is inserted 154 bp 5' to the start of exon 11 and is flanked by a 13- to 17-bp target site duplication. The L1 insertion is present in four generations of the patient's family. The maternal grandfather who carries the insertion does not have hemophilia A, indicating that the insertion is not the cause of hemophilia A in the patient. We have sequenced this insertion and two previously reported de novo L1 insertions in the factor VIII gene in patients JH-27 (3785 bp) and JH-28 (2132 bp). The three nucleotide sequences differ by 0.2-0.8%. All three of these L1 insertions have open reading frames (ORFs) (1192, 642, and 157 aa) and the three derived amino acid sequences are 98-99% identical. The previously reported sequence similarity between L1 3' ORFs and the polymerase domain of reverse transcriptases is maintained in the ORFs of the JH-27 and JH-28 L1 insertions. The presence of open reading frames and the close sequence similarity of these recently inserted L1 elements provide indirect evidence for the existence of a set of functional L1 elements that encode one or more proteins necessary for their retrotransposition.

Base Sequence↗
PubMed1989

In vitro galactosylation of a 110-kDa glycoprotein by an endogenous cell surface galactosyltransferase correlates with the invasiveness of adrenal carcinoma cells.

We have examined the role of a cell surface galactosyltransferase, laminin, and laminin-binding protein (receptor) in the invasion of clonal derivatives of a murine adrenal carcinoma cell line. Although a 10-fold variation was found in the ability to invade a reconstituted basement membrane matrix, levels of intracellular laminin and the laminin-binding protein were shown to be present and secreted equally in all lines. Of the eight lines tested, seven showed a correlation between invasion and the incorporation of [3H]galactose from UDP-[3H]galactose into a 90- to 110-kDa protein. One noninvasive line (clone HSR), however, retained high galactosyltransferase activity yet could not galactosylate the endogenous 90- to 110-kDa substrate. Interestingly, this clone was unable to attach to laminin. Although high galactosyltransferase activity can be consistent with cells of high invasiveness, our results suggest that the galactosylation status of a 90- to 110-kDa Y1 cell surface glycoprotein is most indicative of invasion potential.

Adrenal Gland Neoplasms↗
PubMed1988

Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man.

L1 sequences are a human-specific family of long, interspersed, repetitive elements, present as approximately 10(5) copies dispersed throughout the genome. The full-length L1 sequence is 6.1 kilobases, but the majority of L1 elements are truncated at the 5' end, resulting in a fivefold higher copy number of 3' sequences. The nucleotide sequence of L1 elements includes an A-rich 3' end and two long open reading frames (orf-1 and orf-2), the second of which encodes a potential polypeptide having sequence homology with the reverse transcriptases. This structure suggests that L1 elements represent a class of non-viral retrotransposons. A number of L1 complementary DNAs, including a nearly full-length element, have been isolated from an undifferentiated teratocarcinoma cell line. We now report insertions of L1 elements into exon 14 of the factor VIII gene in two of 240 unrelated patients with haemophilia A. Both of these insertions (3.8 and 2.3 kilobases respectively) contain 3' portions of the L1 sequence, including the poly (A) tract, and create target site duplications of at least 12 and 13 nucleotides of the factor VIII gene. In addition, their 3'-trailer sequences following orf-2 are nearly identical to the consensus sequence of L1 cDNAs (ref. 6). These results indicate that certain L1 sequences in man can be dispersed, presumably by an RNA intermediate, and cause disease by insertional mutation.

Base Sequence↗
PubMed1979

DNA restriction endonuclease analysis for localization of human beta- and delta-globin genes on chromosome 11.

DNA from a clone of a mouse-human hybrid that retained a human chromosome consisting of the major part of chromosome 11 and region q25-26-qter of the X chromosome was digested with various restriction endonucleases, subjected to electrophoresis in agarose gels, and transferred to nitrocellulose filters. The restriction digest pattern of the clone, when hybridized with a 32P-labeled plasmid fragment containing human beta-globin gene sequences, was a composite of the normal human and mouse (A9) patterns. When back-selected in 6-thioguanine to eliminate the 11 translocation chromosome, the hybrid cells showed only the A9 restriction pattern. These results substantiate the localization of beta- and delta-globin genes to human chromosome 11 and exclude the region 11q23-qter as the site.

Animals↗
PubMed1979

A molecular basis for hemoglobin-H disease in American blacks.

We have applied gene counting and restriction endonuclease mapping techniques to the study of two American black families in which there were one or more cases of HbH disease. We found deletions of three of the four normal alpha-globin genes in individuals with HbH disease. In two of these individuals, the chromosome containing the single alpha gene could have originated by crossing over between mispaired alpha genes, resulting in a deletion of about 4.2 kilobases (kb).

Black People↗
PubMed1978

The proportion of all point mutations which are unacceptable: an estimate based on hemoglobin amino acid and nucleotide sequences.

Statistical analysis of the distribution of 156 kinds of human hemoglobin beta (Hbbeta) chain variants suggests that mutations are essentially random in their location. Thus differential fitness, not differential mutability, is the principal source of nonrandom distribution of interspecies differences in Hbbeta amino acid sequence. Similar analyses of both the location and the kind of interspecies differences detected among primates support this viewpoint and lead us to estimate that at least 95% of all amino acid subsitutions,i.e., nonsynonymous mutations, in Hbbeta are functionally unacceptable in homozygous state. Through the combined use of this estimate and the number of nonsynonymous and synonymous substitutions per nucleotide site inferred from comparisons of entire human and rabbit HbbetamRNA nucleotide sequences, we calculate (a) approximately 70% of synonymous Hbbeta mutations are adaptively undersirable and (b) the mutation rate underlying all changes is lesser than or equal to 10(-8) nucleotide substitutions per nucleotide site per year. Apart from such calculations, analyses of nucleotide patterns in HbbetamRNA as well as in rat preproinsulin mRNA reinforce the notion that a large portion of synonymous mutations are functionally unacceptable and rendered so by selective constraint, at a pretranslational level, of the abundance of particular nucleotide doublets such as CpG.

Amino Acid Sequence↗
PubMed1977

The phylogenetic distribution of red cell 2,3 diphosphoglycerate and its interaction with mammalian hemoglobins.

In order to better understand the extent to which 2,3 diphosphoglycerate (DPG) contributes to red cell function in the mammals, we assayed DPG levels in blood from a taxonomically diverse set of 71 species representing 14 orders. In addition, for 66 species and 4 hemoglobin phenotypes of the sheep, the effect of DPG on oxygen affinity was measured by determining P 50 values for hemoglobin in the absence of DPG and at 0.2 mM and 1.0 mM concentrations. Most mammals had high levels of red cell DPG and phosphate-free hemoglobins with a relatively high oxygen affinity. In contrast, two taxonomically unrelated groups had both very low intra-erythrocytic DPG concentrations as well as hemoglobins of native low oxygen affinity that interacted weakly with DPG. This latter group includes the Feloidea (order Carnivora) and the Bovoidea (order Artiodactyla). The relationship between DPG concentration, hemoglobin oxygen affinity and the interaction of DPG with hemoglobin is treated quantitatively to provide a model of mammalian red cell function. This derived expression is compared with descriptive allometric equations for whole blood P 50 and is shown to provide statistically reasonable predictions.

Animals↗
PubMed1976

Functional aspects of hemoglobin evolution in the mammals.

Comparative studies of red cells 2, 3 Diphosphoglycerate (DPG) and its effect on hemoglobin oxygen affinity from a taxonomically diverse set of mammals indicate two anomalous groups: members of the superfamilies Bovoidea (Actiodactyla) and Feloidea (Carnivora). In both taxa all of the individuals assayed had very low or unmeasurable quantities of DPG and red cell lysates with little, if any, DPG effect as measured by the change in oxygen affinity in the absence and presence of the phosphate. However, in both groups compensatory changes have occurred in hemoglobin structure and function so as to reduce the native oxygen affinity and thus cause them to resemble the hemoglobins of DPG-utilizing mammals as they occur in the setting of the red cell. We conclude that this parallelism of function is the result of convergent evolution.

Amino Acid Sequence↗