The complete 186,000 base-pair (bp) human factor VIII gene has been isolated and consists of 26 exons ranging in size from 69 to 3,106 bp and introns as large as 32.4 kilobases (kb). Nine kb of mRNA ...and protein-coding DNA has been sequenced and the mRNA termini have been mapped. The relationship between internal duplications in factor VIII and evolution of the gene is discussed.
Heterochromatin, constitutively condensed chromosomal material, is widespread among eukaryotes but incompletely characterized at the nucleotide level. We have sequenced and analyzed 2.1 megabases ...(Mb) of
Arabidopsis thaliana chromosome 4 that includes 0.5–0.7 Mb of isolated heterochromatin that resembles the chromosomal knobs described by Barbara McClintock in maize. This isolated region has a low density of expressed genes, low levels of recombination and a low incidence of genetrap insertion. Satellite repeats were absent, but tandem arrays of long repeats and many transposons were found. Methylation of these sequences was dependent on chromatin remodeling. Clustered repeats were associated with condensed chromosomal domains elsewhere. The complete sequence of a heterochromatic island provides an opportunity to study sequence determinants of chromosome condensation.
The partial amino-acid sequence of purified human transforming growth factor-beta (TGF-beta) was used to identify a series of cDNA clones encoding the protein. The cDNA sequence indicates that the ...112-amino acid monomeric form of the natural TGF-beta homodimer is derived proteolytically from a much longer precursor polypeptide which may be secreted. TGF-beta messenger RNA is synthesized in various normal and transformed cells.
Amino acid sequences deduced from rat complementary DNA clones encoding the insulin-like growth factor II (IGF-II) receptor closely resemble those of the bovine cation-independent mannose-6-phosphate ...receptor (Man-6-P receptor$^{\text{CI}}$), suggesting they are identical structures. It is also shown that IGF-II receptors are adsorbed by immobilized pentamannosyl-6-phosphate and are specifically eluted with Man-6-P. Furthermore, Man-6-P specifically increases by about two times the apparent affinity of the purified rat placental receptor for $^{125}$I-labeled IGF-II. These results indicate that the type II IGF receptor contains cooperative, high-affinity binding sites for both IGF-II and Man-6-P--containing proteins.
A role for proto-oncogenes in the regulation and modulation of cell proliferation has been suggested by the findings that the B-chain of platelet-derived growth factor (PDGF) is encoded by the ...proto-oncogene sis and that the erb-B oncogene product is a truncated form of the epidermal growth factor (EGF) receptor. Furthermore, the product of the proto-oncogene fms (c-fms) may be related or identical to the receptor for macrophage colony-stimulating factor (CSF-1). v-fms is the transforming gene of the McDonough strain of feline sarcoma virus (SM-FeSV) and belongs to the family of src-related oncogenes which have tyrosine-specific kinase activity. Furthermore, nucleotide sequence analysis of the v-fms gene product revealed topological properties of a cell-surface receptor protein. To elucidate the features involved in the conversion of a normal cell-surface receptor gene into an oncogenic one, we have now determined the complete nucleotide sequence of a human c-fms complementary DNA. The 972-amino-acid c-fms protein has an extracellular domain, a membrane-spanning region, and a cytoplasmic tyrosine protein kinase domain. Comparison of the feline v-fms and human c-fms sequences reveals that the proteins share extensive homology but have different carboxyl termini.
DNA comprising 219 447 bp was sequenced in nine cosmids and verified at >99.9% precision. Of the standard repetitive elements, 187 Alus make up 20.6% of the sequence, but there were only 27 MERs ...(2.9%) and 17 L1 fragments (1.6%). This may be characteristic of such high GC (57%) regions. The sequence also includes an 11.3 kb tract duplicated with 99.2% identity at a distance of 38 kb. The region is 80–90% transcribed and 12.5% translated. Thirteen known genes and their exon-intron borders are all accurately predicted at least in part by GRAIL programs, as are six additional genes. From centromere to telomere, the orientation of transcription varies among the first eight genes, then runs centromeric to telomeric for the next five, and is in the opposite sense for the last six. Eighteen of the 19 genes are associated with CpG islands. Two islands are exact copies in the 11.3 kb repeat units, and could thus give rise to double dosage levels of an X-linked gene. Another island is associated with two genes transcribed in opposite directions. From the sequence data, three genes and their exon structure are inferred. One of them, previously associated with HEX2, is shown to be a different gene unrelated to hexokinases; a second gene, previously known by an EST, is plexin, from its 65.5% identity with the Xenopus analog; and a third is a subunit of a vacuolar H-ATPase, and is named VATPS1.
GPC3,the gene modified in the Simpson–Golabi–Behmel gigantism/overgrowth syndrome (SGBS), is shown to span more than 500 kb of genomic sequence, with the transcript beginning 197 bp 5′ of the ...translational start site. The Xq26.1 region containingGPC3as the only known gene has been extended to >900 kb by sequence analysis of flanking BAC clones. Two GC isochores (40.6 and 42.6% GC) are observed at the 5′ and 3′ ends of the locus, with a large repertoire of repetitive sequences that includes an unusual cluster of four L1 elements >92% identical over 2.8 kb. Eight exons, accounting for the full 2.4-kbGPC3cDNA, have been sequenced along with neighboring intronic regions. PCR assays have been developed to amplify each exon and exon/intron junction sequence, to help discriminate instances of SGBS among individuals with overgrowth syndromes and to facilitate mutational analysis of lesions in the gene.
The most common inherited bleeding disorder in man, haemophilia A, is caused by defect in factor VIII, a component in the blood coagulation pathway. The X-chromosome-linked disease almost certainly ...stems from a heterogeneous collection of genetic lesions. Because, without proper treatment, haemophilia can be a fatal disease, new mutations are necessary to account for its constant frequency in the population. In addition, haemophilia A displays a wide range of severity, and some 15% of haemophiliacs generate high levels of antibodies against factor VIII ('inhibitor patients'). The present work elucidates the molecular genetic basis of haemophilia in some individuals. Using the recently cloned factor VIII gene as a probe, we have identified two different nonsense point mutations in the factor VIII gene of haemophiliacs, as well as two different partial deletions of the gene. Our survey of 92 haemophiliacs indicates no firm correlation between antibody (inhibitor) production and gross gene defects.
Recently, we have shown that mutations in the X-linked glypican 3 (
GPC3) gene cause the Simpson–Golabi–Behmel overgrowth syndrome (SGBS;
Pilia et al., 1996). The next centromeric gene detected is ...another glypican, glypican 4 (
GPC4), with its 5′ end 120 763
bp downstream of the 3′ terminus of
GPC3. One recovered
GPC4 cDNA with an open reading frame of 1668
nt encodes a putative protein containing three heparan sulfate glycosylation signals and the 14 signature cysteines of the glypican family. This protein is 94.3% identical to mouse GPC4 and 26% identical to human GPC3. In contrast to
GPC3, which produces a single transcript of 2.3
kb and is stringently restricted in expression to predominantly mesoderm-derived tissues, Northern analyses show that
GPC4 produces two transcripts, 3.4 and 4.6
kb, which are very widely expressed (though at a much higher level in fetal lung and kidney). Interestingly, of 20 SGBS patients who showed deletions in
GPC3, one was also deleted for part of
GPC4. Thus,
GPC4 is not required for human viability, even in the absence of
GPC3. This patient shows a complex phenotype, including the unusual feature of hydrocephalus; but because an uncle with SGBS is less affected, it remains unclear whether the
GPC4 deletion itself contributes to the phenotype.
Ordered shotgun sequencing (OSS) has been successfully carried out with an Xq25 YAC substrate. yWXD703 DNA was subcloned into λ phage and sequences of insert ends of the λ subclones were used to ...generate a map to select a minimum tiling path of clones to be completely sequenced. The sequence of 135 038 nt contains the entire ANT2 cDNA as well as four other candidates suggested by computer-assisted analyses. One of the putative genes is homologous to a gene implicated in Graves' disease and it, ANT2 and two others are confirmed by EST matches. The results suggest that OSS can be applied to YACs in accord with earlier simulations and further indicate that the sequence of the YAC accurately reflects the sequence of uncloned human DNA.
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