Genomic organization of the human interleukin-12 receptor β2-chain gene

 The interleukin-12 receptor (IL-12R) is composed of two subunits, referred to as β1 and β2. Both chains are necessary for high-affinity IL-12 binding and signalling, although only the IL-12Rβ2 chain contains the intracellular tyrosine residues responsible for STAT4 activation. This study presents the intron-exon organization of the human IL-12Rβ2-chain gene. Polymerase chain reaction (PCR) primers designed across the cDNA (U46198) were used to trace introns, by comparing PCR product sizes obtained using cDNA and genomic DNA as templates. PCR products spanning introns were sequenced to determine the exact splice sites and flanking regions. The coding region of the gene was found to consist of 15 exons and 14 introns. All intron-exon boundaries are consistent with the consensus sequence for splice junctions (5′ GT/AG 3′). Comparison of the intron-exon organization with the human GCSFR gene indicated a remarkably well conserved genomic organization between these two class I cytokine receptors. Interestingly, we identified an alternatively spliced mRNA, encoding a putative, truncated protein, lacking all signalling potential. Received: 21 July 1999 / Revised: 2 September 1999     

The gene encoding the large human neurofilament subunit (NF-H) maps to the q121–q131 region on human chromosome 22

Summary Using a rat cDNA probe encoding for the C-terminal textension of the large neurofilament subunit (NF-H), we have assigned, by in situ hybridization, the human NF-H gene to the q121–q131 region of chromosome 22. This localization may have implications in neurological diseases such as meningioma where a recessive locus involved in oncogenesis is located within this region.     

Bovine αs1-casein gene sequences direct high level expression of human granulocyte-macrophage colony-stimulating factor in the milk of transgenic mice

The generation is reported of transgenic mice expressing human granulocyte-macrophage colony-stimulating factor (GM-CSF) or human erythropoietin (EPO) under the control of bovine s1- casein regulatory sequences. GM-CSF expression was specific to the mammary gland, and levels of human GM-CSF in transgenic mouse milk were in the range of mg ml–1. The specific activity of the milk GM-CSF was similar to that of the recombinant protein produced in Escherichia coli, and the glycosylation-derived size heterogeneity corresponded to that of the native human protein. In spite of the identical bovine regulatory sequences of the fusion genes, the levels of human EPO in transgenic mouse milk were 103--106 times lower than those of GM-CSF, ranging from 0.003 to 3 g ml–1. There appeared to be a positive correlation between the amount of EPO in the milk of lactating females and blood haematocrit values. In view of this, other type of constructs should be used to achieve more efficient EPO expression and to circumvent concomitantly-occurring adverse effects. In contrast, the high-level production of recombinant GM-CSF, its resemblance to the native mammalian protein, and mild adverse consequences of transgene expression imply that the current construct could be used for generation of larger GM-CSF transgenic anim als to produce this protein in quantities sufficient for therapeutic purposes     

Organization of the human tumour necrosis factor receptor-associated factor 1 (TRAF1) gene and mapping to chromosome 9q33–34

A new family of signal transducing proteins, associated with members of the tumour necrosis factor receptor (TNFR) superfamily, has recently been identified. The structural hallmark of these molecules is a novel C-terminal homology region of 230 bp designated as TRAF (TNF receptor-associated factor) domain, which is involved in a variety of specific protein–protein interactions. To elucidate the human TRAF1 gene structure for identification of potential regulatory elements, a set of genomic polymerase chain reaction (PCR) fragments was generated, which comprised the whole coding region of TRAF1. These fragments were cloned and partially sequenced to map splicing sites. The human TRAF1 gene was found to have a total length of approx. 12 kb. It is split into six exons, four of which encode for parts of the TRAF domain. Analysis of the genomic structure of the TRAF domains of human TRAF2 and 3 suggests that these domains are also encoded by several exons. The putative promotor region of the TRAF1 gene was isolated by use of a PCR-based genomic walking approach. Fluorescence in situ hybridization was used to map this gene to chromosome 9q33–34.     

Chromosomal locations of the gene coding for the CD3 (T3) γ subunit of the human and mouse CD3/T-cell antigen receptor complexes

The gene coding for the M _r 26000 chain of the human CD3 (T3) antigen/T-cell antigen receptor complex was mapped to chromosome band 11q23 by using a cDNA clone (pJ6T3 -2), by in situ hybridization to metaphase chromosomes and by Southern blot analysis of a panel of human-rodent somatic cell hybrids. The mouse homolog, here termed Cdg-3, was mapped to chromosome 9 using the mouse cDNA clone pB10.AT3 -1 and a panel of mouse-hamster somatic cell hybrids. Similar locations for the CD3 genes have been described previously. Thus, the corporate results indicate that the CD3 and genes have remained together since they duplicated about 200 million years ago.     

Detection of a CfoI polymorphism within exon 5 of the human neuronal nicotinic acetylcholine receptor α4 subunit gene (CHRNA4)

The gene coding for the 4 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA4) has been recently mapped in the candidate region for benign familial neonatal convulsions (BFNC) on chromosome 20q13.2–q13.3. The region is only partially covered with polymorphic markers, and so far no PCR-based polymorphisms have been described in the critical region for BFNC. We now report the first polymorphic marker in the coding region of CHRNA4. The new marker, which is detected by PCR, will be useful for evaluation of the role of CHRNA4 as a candidate gene for BFNC. It will further enable the investigation of this important brain-specific gene in association studies with different types of epileptic diseases and other neurological disorders.     

Cloning and chromosomal assignment of the bovine interleukin-2 receptor alpha (IL-2Rα) gene

The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number U24226.     

Assignment of the human γ-crystallin gene cluster (CRYG) to the long arm of chromosome 2, region q33–36

Summary The -crystallins of the human eye lens are encoded by a multigene family of which at least six genes have recently been assigned to chromosome 2. We have now localized these genes to the distal region of the long arm of chromosome 2 (region q33-36, most probably q34-35) using somatic cell hybrids containing different parts of this chromosome and by in situ hybridization. The -crystallin genes map to the same chromosomal region as IDH-1. Similar linkage exists between the loci Len-1 and Idh-1 on mouse chromosome 1.     

Regional assignment of the human uroporphyrinogen III synthase (UROS) gene to chromosome 10q25.2→q26.3

Summary Uroporphyrinogen III synthase [UROS; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75] is the fourth enzyme in the human heme biosynthetic pathway. The recent isolation of the cDNA encoding human UROS facilitated its chromosomal localization. Human UROS sequences were specifically amplified by the polymerase chain reaction (PCR) from genomic DNA of two independent panels of human-rodent somatic cell hybrids. There was 100% concordance for the presence of the human UROS PCR product and human chromosome 10. For each of the other chromosomes, there was 19%–53% discordance with human UROS. The chromosomal assignment was confirmed by Southern hybridization analysis of DNA from somatic cell hybrids with the full-length UROS cDNA. Using human-rodent hybrids containing different portions of human chromosome 10, we assigned the UROS gene to the region 10q25.2 q26.3.     

Assignment of the prealbumin (PALB) gene (familial amyloidotic polyneuropathy) to human chromosome region 18q11.2–q12.1

Summary The assignment of the human prealbumin (PALB) gene to chromosome region 18q11–q12.1 has been achieved using a human genomic probe in the study of human-mouse somatic cell hybrids and by in situ hybridization. Because familial amyloidotic polyneuropathy was reported previously to be due to a mutation in prealbumin, it can be inferred that the gene for this disorder also maps to 18q11.2–q12.1.     

A 3,387?bp 5′-flanking sequence of the goat alpha-S1-casein gene provides correct tissue-specific expression of human granulocyte colony-stimulating factor (hG-CSF) in the mammary gland of transgenic mice

A new expression vector containing the 1,944 bp 5'-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3'-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19-40 μg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.     

Mapping and genomic characterization of the gene encoding diacylglycerol kinase γ (DAGK3): assessment of its role in dominant optic atrophy (OPA1)

The family of diacylglycerol kinases (DAGKs) is known to play an important role in signal transduction linked to phospholipid turnover. In the fruitfly Drosophila melanogaster, a human DAGK ortholog, DGK2, was shown to underlie the phenotype of the visual mutant retinal degeneration A (rdgA). Previously, the gene encoding a novel member of the human DAGK family, termed DAGK3, was cloned and demonstrated to be abundantly expressed in the human retina. Based on these findings we reasoned that DAGK3 might be an excellent candidate gene for a human eye disease. In the present study, we report the genomic organization of the human DAGK3 gene, which spans over 30 kb of genomic DNA interrupted by 23 introns. In addition, we have mapped the gene locus by fluorescence in situ hybridization to 3q27–28, overlapping the chromosomal region known to contain the gene underlying dominant optic atrophy (OPA1), the most common form of hereditary atrophy of the optic nerve. Mutational analysis of the entire coding region of DAGK3 in 19 unrelated German OPA1 patients has not revealed any disease-causing mutations, therefore excluding DAGK3 as a major cause underlying OPA1. Received: 24 August 1998 / Accepted: 13 October 1998     

Identification of a putative DNA replication origin in the λ-aminobutyric acid receptor subunit β3 and α5 gene cluster on human chromosome 15q11-q13, a region associated with parental imprinting and allele-specific replication timing

The region containing the GABA_A receptor β3 and α5 subunit-encoding genes is subject to parental imprinting and is organized in different allele-specific replication timing domains. A 60-kb domain displaying a maternal early/paternal late pattern of allele-specific replication timing asynchrony is nested within a larger region displaying the opposite pattern. The proximal portion of this maternal early replicating domain is incorporated into phage clone λ84. In order to identify DNA structures which may be associated with the boundary between the replication domains, phage λ84 has been subcloned into smaller fragments and several of these have been analyzed by nucleotide sequencing. A plot of helical stability for 13 kb of contiguous sequence reveals several A +T-rich regions which display potential DNA unwinding. The plasmid subclones from phage λ84 have been analyzed for bent DNA and one of these, p82, contains bent DNA and overlaps with the region of highest potential helical instability. Of the seven plasmids tested, only p82 shows strong autonomous replication activity in an in vitro replication assay, with replication initiating within the genomic insert. These results suggest that a putative origin of DNA replication contained within p82 may play a role in establishing the allele-specific replication timing domains in the GABA_A receptor subunit gene cluster.