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J P Hjorth

Publications and source records attributed to J P Hjorth.

At least 19 recordsLinked to original sources
PubMed2001

Protein-binding elements in the proximal parotid secretory protein gene enhancer essential for salivary-gland-specific expression.

The murine parotid secretory protein (PSP) gene is expressed at high levels in the parotid gland and at lower levels in the sublingual gland. A proximal enhancer core necessary for tissue-specific expression was identified previously, and it was demonstrated that one element, parotid gland element I (PGE I), exhibited specific binding of parotid gland nuclear proteins. In the present study, we demonstrate that a related adjacent element, PGE II, which binds nuclear proteins in a much less tissue-restricted manner, is able to compete with PGE I for binding of parotid-gland-specific factors. The functional significance of PGE I and PGE II was examined in transgenic mice. Deletion of PGE II reduced transgene expression only in the parotid gland, whereas deletion of PGE I appeared to reduce expression in both of the PSP-expressing salivary glands. Combined deletion of PGE I and PGE II reduced expression below the limit of detection. Thus PGE I and PGE II are functionally important salivary-gland-specific binding elements that are necessary for the salivary-gland-specific expression of a PSP minigene in transgenic mice.

Animals↗
PubMed2001

Pigs expressing salivary phytase produce low-phosphorus manure.

To address the problem of manure-based environmental pollution in the pork industry, we have developed the phytase transgenic pig. The saliva of these pigs contains the enzyme phytase, which allows the pigs to digest the phosphorus in phytate, the most abundant source of phosphorus in the pig diet. Without this enzyme, phytate phosphorus passes undigested into manure to become the single most important manure pollutant of pork production. We show here that salivary phytase provides essentially complete digestion of dietary phytate phosphorus, relieves the requirement for inorganic phosphate supplements, and reduces fecal phosphorus output by up to 75%. These pigs offer a unique biological approach to the management of phosphorus nutrition and environmental pollution in the pork industry.

6-Phytase↗
PubMed2000

A minimal c-fes cassette directs myeloid-specific expression in transgenic mice.

The c-fes proto-oncogene encodes a 92-kd protein tyrosine kinase whose expression is restricted largely to myeloid and endothelial cells in adult mammals. A 13.2-kilobase (kb) human c-fes genomic fragment was previously shown to contain cis-acting element(s) sufficient for a locus control function in bone marrow macrophages. Locus control regions (LCRs) confer transgene expression in mice that is integration site independent, copy number dependent, and similar to endogenous murine messenger RNA levels. To identify sequences required for this LCR, c-fes transgenes were analyzed in mice. Myeloid-cell-specific, deoxyribonuclease-I-hypersensitive sites localized to the 3' boundary of exon 1 and intron 3 are required to confer high-level transgene expression comparable to endogenous c-fes, independent of integration site. We define a minimal LCR element as DNA sequences (nucleotides +28 to +2523 relative to the transcription start site) located within intron 1 to intron 3 of the human locus. When this 2.5-kb DNA fragment was linked to a c-fes complementary DNA regulated by its own 446-base-pair promoter, integration-site-independent, copy-number-dependent transcription was observed in myeloid cells in transgenic mice. Furthermore, this 2.5-kb cassette directed expression of a heterologous gene (enhanced green fluorescent protein) exclusively in myeloid cells. The c-fes regulatory unit represents a novel reagent for targeting gene expression to macrophages and neutrophils in transgenic mice.

Animals↗
PubMed1999

A humanized model for multiple sclerosis using HLA-DR2 and a human T-cell receptor.

Multiple sclerosis (MS) is a complex chronic neurologic disease with a suspected autoimmune pathogenesis. Although there is evidence that the development of MS is determined by both environmental influences and genes, these factors are largely undefined, except for major histocompatibility (MHC) genes. Linkage analyses and association studies have shown that susceptibility to MS is associated with genes in the human histocompatibility leukocyte antigens (HLA) class II region, but the contribution of these genes to MS disease development less compared with their contribution to disorders such as insulin-dependent diabetes mellitus. Due to the strong linkage disequilibrium in the MHC class II region, it has not been possible to determine which gene(s) is responsible for the genetic predisposition. In transgenic mice, we have expressed three human components involved in T-cell recognition of an MS-relevant autoantigen presented by the HLA-DR2 molecule: DRA*0101/DRB1*1501 (HLA-DR2), an MHC class II candidate MS susceptibility genes found in individuals of European descent; a T-cell receptor (TCR) from an MS-patient-derived T-cell clone specific for the HLA-DR2 bound immunodominant myelin basic protein (MBP) 4102 peptide; and the human CD4 coreceptor. The amino acid sequence of the MBP 84-102 peptide is the same in both human and mouse MBP. Following administration of the MBP peptide, together with adjuvant and pertussis toxin, transgenic mice developed focal CNS inflammation and demyelination that led to clinical manifestations and disease courses resembling those seen in MS. Spontaneous disease was observed in 4% of mice. When DR2 and TCR double-transgenic mice were backcrossed twice to Rag2 (for recombination-activating gene 2)-deficient mice, the incidence of spontaneous disease increased, demonstrating that T cells specific for the HLA-DR2 bound MBP peptide are sufficient and necessary for development of disease. Our study provides evidence that HLA-DR2 can mediate both induced and spontaneous disease resembling MS by presenting an MBP self-peptide to T cells.

Animals↗
PubMed1998

Novel salivary gland specific binding elements located in the PSP proximal enhancer core.

The murine parotid secretory protein (PSP) gene is expressed selectively at high levels in parotid and sublingual salivary glands. Previously, the transcriptional activity of a PSP mini-gene, called Lama, was shown to be dependent on a 1.5 kb region located 3 kb upstream of the transcription start site. Here, functional studies in transgenic mice demonstrate that this proximal regulatory region has properties of a parotid and sublingual gland specific enhancer. Protein-binding experiments identify multiple sequence-specific binding complexes spanning the entire 1.5 kb enhancer region. Several sequence elements bound specifically by parotid and/or sublingual gland nuclear extracts, including consensus binding elements for previously described transcription factors as well as novel binding elements are located in the proximal enhancer region. A deletion analysis of the enhancer region in transgenic mice identified a core sequence of 700 bp. This region contains five elements bound specifically by nuclear proteins isolated from the PSP-expressing parotid and sublingual glands. Two of these elements, denoted parotid gland element I (PGE I) and sublingual gland element I (SLE I), are novel salivary gland specific binding elements, bound uniquely by parotid and sublingual gland nuclear extracts, respectively.

Amino Acid Sequence↗
PubMed1998

Allele-specific expression of the PSP gene in the mouse sublingual glands.

There are two known alleles of the mouse parotid secretory protein (PSP) gene: Pspa and Pspb. Pspa is carried by DBA/2J mice and Pspb is carried by C57BL/6J mice. Eighty-eight mice derived from a F1(C57BL/6J x DBA/2J) to DBA/2J backcross were analysed for PSP mRNA expression in the sublingual glands. Expression was found in heterozygous mice only. This indicates that only Pspb is expressed in this tissue. Furthermore, it maps the allele-specific sublingual gland determinant within 3.4 cM of Psp. Previous analysis of Pspb identified an enhancer-like region in position -4.6 to -3.1 kb that was necessary for transgene expression in the sublingual glands. Here it is shown that the corresponding region in Pspa enhances transgene expression in the sublingual glands as efficiently. The implications for regulation of PSP mRNA expression in the sublingual glands are discussed.

Alleles↗
PubMed1998

The main regulatory region in the murine PSP gene is a parotid gland enhancer.

The murine PSP gene is expressed at a high-level in the parotid glands. To extend the knowledge of parotid gland expression and develop tools for expression of heterologous proteins in this tissue, the regulation of the PSP gene was studied using transgenic mice. High-level parotid gland expression of the PSP gene was indicated to depend on a novel regulatory region situated between -8.0 and -6.5 kb. Together with previous results this indicates that the main regulatory elements in the PSP gene are situated between -8.0 to -3.1 kb. This region was shown to activate a heterologus SV40 early promoter in the parotid glands of transgenic mice, suggesting that the PSP gene is controlled by enhancer sequences. A novel Psp derived 9.7 kb parotid gland expression cassette, Lama IV, carrying all known regulatory regions in the PSP gene was expressed at high-levels in the parotid glands and should prove highly useful for expression of heterologous proteins in the saliva of transgenic mice.

Animals↗
PubMed1997

A cassette for high-level expression in the mouse salivary glands.

Expression in the mouse salivary glands may be used as a model system for studies involving oral cavity delivery of gene products. Previously, sequences from the mouse Psp gene were used to build a minigene construct denoted 'Lama'. This construct was used as a cassette for expression of human factor VIII light chain in mouse saliva. However, whereas the endogenous Psp mRNA is the most abundant protein-coding transcript in the parotid glands, the Lama mRNA was expressed below 1% of the level of Psp mRNA in these glands. Here, we show that a 25-kb cosmid-derived DNA fragment (PspX25) carrying the structural gene and large flanking areas of Psp is expressed in all 14 analysed lines in the parotid glands. The average level of transgene expression was estimated to be 45% of that of the endogenous Psp gene. More importantly, it was possible to transfer PspX25's ability for high-level parotid gland expression to the Lama construct.

Animals↗
PubMed1994

High-level salivary gland expression in transgenic mice.

A 7.1 kb mini-gene construct containing cloned DNA from the murine parotid secretory protein (PSP) gene with 6.2 kb of the promoter, has previously been shown to direct specific mRNA expression to the salivary glands in transgenic mice. However, the level of transgene expression in the parotid gland was only a few percent of the endogenous level. This indicated that elements necessary for high-level expression are still to be found. In this study, we have searched for such regulatory elements in additional flanking regions by using a 25 kb cloned Pspb fragment containing the complete structural gene, 11.4 kb of 5'-flanking sequence, and 2.5 kb 3'-flanking sequence as a transgene. To distinguish the expression of the transgene from that of the endogenous gene, we took advantage of an allelic difference, using an oligonucleotide that recognized the mRNA from Pspb and the transgene but not that from the other allele, Pspa. The expression of the transgene was examined in animals homozygous for Pspa. Three independent integrations all exhibited a level of parotid-gland-specific expression that corresponded to that of the endogenous gene. Thus, sequences responsible for this high-level PSP mRNA expression are situated within the genomic DNA of the transgene.

Alleles↗
PubMed1992

Tissue-specific expression in the salivary glands of transgenic mice.

Using a DNA construct, named Lama, derived from the murine parotid secretory protein (PSP) gene, we have obtained salivary gland specific gene expression in transgenic mice. Lama is a PSP minigene and allows analysis of the PSP gene 5' regulatory region by transgenesis. We show here that the regulatory region included in Lama with 4.6 kb of 5' flanking sequence is sufficient to direct expression specifically to the salivary glands. The expression level in the parotid gland is only about one percent of the PSP mRNA level, while that of the sublingual gland is near the PSP mRNA level. This suggests significant differences in the PSP gene regulation in the two glands. In addition, Lama is a secretory expression vector in which cDNAs or genomic fragments can be inserted. We demonstrate that the Lama construct can direct the expression of a heterologous cDNA encoding the C-terminal peptide of human factor VIII to salivary glands and that the corresponding peptide is secreted into saliva.

Amino Acid Sequence↗
PubMed1992

Expression of a cytomegalovirus IE-1-factor VIII cDNA hybrid gene in transgenic mice.

A construct containing the 5' end of the human cytomegalovirus major immediate early gene fused to the human coagulation factor VIII cDNA was used to produce transgenic mice. Two out of five transgenic lines transcribed the construct. The expression was consistently seen in a limited number of tissues and was highest in muscle tissues. This is in contrast to the almost ubiquitous activity demonstrated in earlier studies with the IE-1 enhancer/promoter. Human factor VIII protein was detected immunochemically in muscle tissues at levels several times higher than in human plasma.

Animals↗
PubMed1990

Retropseudogenes constitute the major part of the human elongation factor 1 alpha gene family.

The elongation factor 1 alpha (EF-1 alpha) is a protein which promotes the GTP-dependent binding of aminoacyl-tRNA to ribosomes in the protein synthesis process. A human gene coding for EF-1 alpha has previously been cloned and sequenced along with a pseudo-gene. Here, we have further analyzed the family of human EF-1 alpha genes. Using an EF-1 alpha cDNA as probe twelve genomic EF-1 alpha-like clones were isolated and analyzed. Four of these were sequenced and found to contain EF-1 alpha retropseudogenes. A Southern blot analysis indicated that the remaining eight clones also contained retropseudogenes. Genomic Southern blot analysis revealed at least twenty loci in the human genome with sequence homology to the EF-1 alpha cDNA. Besides the already described active gene only one potentially active locus was found. The others appeared to be retropseudogenes. EF-1 alpha retropseudogenes were also found to be abundant in the mammalian species mouse and pig, while the chicken contained only one presumably active EF-1 alpha gene.

Blotting, Southern↗
PubMed1989

Cloning and sequencing of the immunoglobulin A1 protease gene (iga) of Haemophilus influenzae serotype b.

Secretion of immunoglobulin A1 (IgA1) proteases is a characteristic of Haemophilus influenzae and several other bacterial pathogens causing infectious diseases, including meningitis. Indirect evidence suggests that the proteases are important virulence factors. In this study, we cloned the iga gene encoding immunoglobulin A1 (IgA1) protease from H. influenzae serotype b into Escherichia coli, in which the recombinant H. influenzae iga gene was expressed and the resulting protease was secreted. Sequencing a part of a 7.5-kilobase DNA fragment containing the iga gene revealed a large open reading frame with a strongly biased codon usage and having the potential of encoding a protein of 1,541 amino acids and a molecular mass of 169 kilodaltons. Putative promoter and terminator elements flanking the open reading frame were identified. Comparison of the deduced amino acid sequence of this H. influenzae IgA1 protease with that of a similar protease from Neisseria gonorrhoeae revealed several domains with a high degree of homology. Analogous to mechanisms known from the N. gonorrhoeae IgA protease secretion, we propose a scheme of posttranslational modifications of the H. influenzae IgA1 protease precursor, leading to a secreted protease with a molecular mass of 108 kilodaltons, which is close to the 100 kilodaltons reported for the mature IgA1 protease.

Amino Acid Sequence↗
PubMed1988

Limited diversity of the immunoglobulin A1 protease gene (iga) among Haemophilus influenzae serotype b strains.

Immunoglobulin A1 (IgA1) proteases are thought to be important virulence factors in certain bacterial infections, including meningitis, and may have potential usage in vaccines. In this study, we compared the locations of EcoRI, BamHI, and PstI restriction endonuclease sites in the IgA1 protease gene (iga) region of whole-cell DNA from 76 Haemophilus influenzae strains. The analysis was performed by using isolated fragments of the cloned iga gene, which encodes the IgA1 protease originating from a H. influenzae serotype d strain, as probes in Southern blot experiments. All strains, including three without detectable IgA1 protease activity, had DNA sequences with a high degree of homology to the iga probes. The numbers and sizes of the DNA fragments hybridizing with the probes indicated that only three strains, none of which was of serotype b, had more than one iga gene. The iga restriction fragment length patterns of 60 clinical isolates of serotype b were of only four distinct types, which correlated with previously observed clusters of multilocus genotypes (electrophoretic types). This correlation supports the concept of the clonal population structure of H. influenzae. Three of the iga gene restriction types, which appear to represent 98% of the H. influenzae serotype b population, encode IgA1 proteases that were inhibited by antisera to any one of these types and therefore could form the basis for the development of a vaccine against H. influenzae meningitis.

Antibodies, Bacterial↗
PubMed1986

Coordination of murine parotid secretory protein and salivary amylase expression.

PSP, parotid secretory protein, and salivary amylase are the major secretory proteins of mouse parotid gland where they appear in a constant ratio. Here we describe the isolation of the PSP gene and show through expression analysis on this and the salivary amylase gene that the two genes are transcribed in a coordinate fashion in adult animals, whereas the activation profiles are different during postnatal development. An explanation is put forward that involves activation of the genes at different stages of the acinar cell differentiation, leading in adults to the maximal and thus proportionate expression.

Amylases↗
PubMed1985

Molecular cloning of mouse PSP mRNA.

PSP is the most abundant translation product of mouse parotid glands where its production is co-ordinated with that of salivary amylase. The synthesis of these two proteins apparently is restricted to this tissue. In order to enable us to study common regulatory elements in the genes of the two proteins, double stranded cDNA, synthesized for parotid gland poly (A)+ RNA, was cloned. DNA sequencing of three clones complementary to the most abundant messenger indicated overlap and resulted in a total sequence of 867 nucleotides. Translation of this sequence revealed that at one end the amino acid sequence was the same as the N-terminal sequence of PSP. The sequence contains 60 nucleotides coding for part of or the complete signal peptide, 645 nucleotides coding for the PSP protein, and 162 nucleotides that apparently are not translated. Southern blot analysis suggests a simple structure for the PSP gene in mouse and man.

Amino Acid Sequence↗