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K Nordqvist

Publications and source records attributed to K Nordqvist.

17 recordsLinked to original sources
PubMed2001

Characterization of the Xp21-23 region in the wood lemming, a region involved in XY sex reversal.

The wood lemming (Myopus schisticolor) harbors two types of X chromosome, a normal X and a variant X, designated X*. The X* chromosome contains a mutation that causes XY sex reversal. We have previously demonstrated that the Xp21-23 region is deleted from X* and is associated with XY sex reversal. To further analyze the deleted region, we have constructed and characterized seven X chromosome- and region-specific recombinant DNA libraries. Further, we have screened mouse fetal gonad cDNA libraries with the microdissected Xp21-23 DNA as a probe in an attempt to identify homologous and expressed sequences from the deletion. Fourteen positive clones were isolated, and sequence analyses showed that ten of these contained identical sequences homologous to mouse gamma-satellite sequences. One of the remaining four was perfectly homologous to the mouse gene Ccth (chaperonin containing t-complex polypeptide 1, eta subunit). Southern blot indicated that the Ccth cDNA was located on the X chromosome, not deleted from the X* but closely linked to the deletion region. Although the role of the Ccth containing region in sex determination of the wood lemming requires additional studies, the isolation of the mouse Ccth gene by the deletion Xp21-23 probe could be important since this gene is mainly expressed in testis.

Animals↗
PubMed2001

The melanoma antigen genes--any clues to their functions in normal tissues?

The melanoma antigen (MAGE) genes were initially isolated from melanomas and turned out to have an almost exclusively tumor-specific expression pattern. This led to the idea of using MAGE genes as targets for cancer immunotherapy, and MAGE peptides are currently being investigated as immunizing agents in clinical studies. Although 23 human and 12 mouse MAGE genes have been isolated in various tumors and characterized, not much is known about their function in normal cells. In adult tissues, most MAGE genes are expressed only in the testis and expression patterns suggest that this gene family is involved in germ cell development. In contrast to the MAGE genes, more functional data have accumulated around the MAGE related gene necdin. This gene encodes a neuron-specific growth suppressor that facilitates the entry of the cell into cell cycle arrest. Necdin is functionally similar to the retinoblastoma protein and binds to and represses the activity of cell-cycle-promoting proteins such as SV40 large T, adenovirus E1A, and the transcription factor E2F. Necdin also interacts with p53 and works in an additive manner to inhibit cell growth. In this review we will focus on the normal functions of MAGE genes and we speculate, based on the patterns of MAGE expression and on observed functions of necdin, that this gene family is involved in cell cycle regulation, especially during germ cell development.

Amino Acid Sequence↗
PubMed2001

Protocol for using signal peptide differential display and representational difference analysis to isolate differentially expressed cDNAs from fetal mouse brain.

Comparing female and male brain structures reveals a variety of sex differences in many vertebrates. Some of these differences are thought to be induced during the fetal period by the effect of steroid hormones produced in the gonads. Not much is known about molecular mechanisms involved in gender-specific development of the brain. We have taken a broad approach to isolate sex-specific genes from 18.5 days post coitum brain (A. Eriksson, C. Wahlestedt and K. Nordqvist. 1999. Isolation of sex-specific cDNAs from fetal mouse brain using mRNA differential display and representational difference analysis. Mol. Brain Res., 74, 91-97). Female and male mouse brains were screened with the signal peptide differential display, developed in our laboratory, and with a modified representational difference analysis of cDNA. The resulting sex-specific fragments were verified by semi-quantitative RT-PCR. Here we describe these methods in detail.

Animals↗
PubMed2000

Mage-b4, a novel melanoma antigen (MAGE) gene specifically expressed during germ cell differentiation.

The MAGE genes were initially isolated from different kinds of tumors, and based on their virtually exclusive tumor-specific expression in adult tissues, they have been used as targets for cancer immunotherapy. However, although a large number of MAGE genes have now been identified and extensively studied in tumors of various origin, their functions in normal cells remain unknown. Here we describe the isolation and characterization of a novel murine MAGE homologue, Mage-b4. mRNA expression studies in a wide variety of adult and embryonic tissues revealed that Mage-b4 is specifically expressed in fetal and adult gonads. An antibody specific to Mage-b4 was developed, and using this antibody, we found that the Mage-b4 protein was confined to the cytoplasm of germ cells. Double-labeling experiments using antibodies against the meiosis-specific SCP3 protein and the Mage-b4 protein showed that Mage-b4 is down-regulated as the germ cells enter meiosis in adult testis. In contrast, Mage-b4 was expressed in female germ cells throughout meiosis, and the protein was also found in dormant primary oocytes.

Amino Acid Sequence↗
PubMed1999

Isolation of sex-specific cDNAs from fetal mouse brain using mRNA differential display and representational difference analysis.

Comparing female and male brain structures reveals a variety of sex differences in many vertebrates. These differences are manifested throughout the brain, in regions such as the hypothalamus, the preoptic area and the amygdala. Some are thought to be induced during the fetal period by the effect of steroid hormones produced in the gonads. It is well-established that fetal androgens, probably through the conversion to estrogen by the enzyme aromatase, masculinize the nervous system and set adult mounting behavior in rodents. However, less is known about molecular mechanisms involved in gender-specific development of the brain. We have taken a broad approach to isolate sex-specific genes from fetal brain. mRNAs from 18.5 days post-coitum (dpc) female and male mouse brain were screened with the classical and the recently developed signal peptide differential display (SPDD) and with representational difference analysis of cDNA (cDNA-RDA). Two sex-specific cDNAs were isolated, F29 and M17, corresponding to the female-specific Xist gene and the male-specific Smcy gene, respectively.

Amino Acid Sequence↗
PubMed1998

Testatin: a cystatin-related gene expressed during early testis development.

To isolate genes involved in morphogenic aspects of testis development, and which may act in cell signaling pathways downstream of the testis-determining gene Sry, we have developed a modified mRNA differential display method named signal peptide differential display. It was used to target those genes that encode proteins having a signal peptide sequence. By using this method, we isolated a gene named testatin. This gene was found to be related to a group of genes that encodes cysteine protease inhibitors known as cystatins. Cystatins and their target proteases have been associated with tumor formation and metastasis, but also are involved in natural tissue remodeling events such as bone resorption and embryo implantation. We show that testatin expression is restricted to fetal gonads and adult testis. Furthermore, testatin is expressed during testis cord formation in pre-Sertoli cells, believed to be the site of Sry action, at a time immediately after the peak of Sry expression. This finding suggests that testatin might be activated by transcription factors that are known to orchestrate the early testis development pathway. This gene therefore represents one of the putative downstream targets likely to have an essential role in tissue reorganization during early testis development.

Amino Acid Sequence↗
PubMed1998

Sry and the testis: molecular pathways of organogenesis.

The gene Sry acts as a switch, initiating pathways leading to the differentiation of a testis rather than an ovary from the indifferent gonad (genital ridge) in mammals. The early events following Sry expression include rapid changes in the topographical organization of cells in the XY gonad. Sry must therefore initiate signaling pathways that direct male-specific patterns of proliferation, migration, cell-cell organization, and vascularization. We have identified an increase in male-specific proliferation by 12.0 days post coitum, while proliferation in the female gonad declines. We have also observed male-specific cell migration from the mesonephros into the gonad in a composite organ culture system in which gonads from wild-type mice (CD1) and mesonephroi from a transgenic strain expressing beta-galactosidase in all its cells (ROSA26) were grafted together in vitro at the indifferent stage of gonadogenesis. Migration depends on an active signal that requires the presence of a Y chromosome in the gonadal portion of the graft. The signals that trigger migration operate over considerable distances, suggesting either a long-range diffusible factor or the involvement of a rapid and efficient relay mechanism. Identification of the somatic cells contributed from the mesonephros with cell-specific markers indicated that some of the migrating cells were endothelial, revealing differences in processes of vascularization between male and female gonads. A second distinct population of migrating cells lay in close apposition to endothelial cells, and a third population occupied positions circumscribing areas of condensing Sertoli cells.

Animals↗
PubMed1997

Male-specific cell migration into the developing gonad.

BACKGROUND: The gene Sry acts as a developmental switch, initiating a pathway of gene activity that leads to the differentiation of testis rather than ovary from the indifferent gonad (genital ridge) in mammalian embryos. The early events following Sry expression include rapid changes in the topographical organization of cells in the XY gonad. To investigate the contribution of mesonephric cells to this process, gonads from wild-type mice (CD1), and mesonephroi from a transgenic strain ubiquitously expressing beta-galactosidase (ROSA26), were grafted together in vitro. After culture, organs were fixed and stained for beta-galactosidase activity to identify cells contributed from the mesonephros to the male or female gonad. RESULTS: Migration of mesonephric cells occurred into XY but not XX gonads from 11.5-16.5 days post coitum (dpc). Somatic cells contributed from the mesonephros were distinguished by their histological location and by available cell-specific markers. Some of the migrating cells were endothelial; a second population occupied positions circumscribing areas of condensing Sertoli cells; and a third population lay in close apposition to endothelial cells. CONCLUSIONS: OFFgration from the mesonephros to the gonad is male specific at this stage of development and depends on an active signal that requires the presence of a Y chromosome in the gonad. The signals that trigger migration operate over considerable distances and behave as chemoattractants. We suggest that migration of cells into the bipotential gonad may have a critical role in initiating the divergence of development towards the testis pathway.

Animals↗
PubMed1997

An mRNA differential display strategy for cloning genes expressed during mouse gonad development.

The mRNA differential display technique has become a popular method for isolating novel genes in a variety of biological systems including carcinogenesis, hormone regulation, plant biology and neurobiology. We have further developed the method by optimizing different steps for the use of small amounts of material, such that differential display can be used in the study of developmental biology. Our techniques include a new assay for elimination of false positive cDNA clones and a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method for the rapid analysis of differences in gene expression. This improved mRNA differential display strategy requires less than 4 microg of total RNA. We have used it for the isolation of genes which are expressed during gonad development in the mouse. One of the cDNAs found, cDNA 4.3 which corresponds to a part of the gene encoding the steroid hydroxylase 3betaHSD I, was shown to be a valuable marker for adrenal development and for Leydig cell differentiation and organization during testis development.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Sex differentiation -- gonadogenesis and novel genes.

During embryogenesis, most organ rudiments differentiate into only one type of organ and functional mutations are normally lethal for the embryo. However, the indifferent gonad has two options, to form either a testis or an ovary, and mutations of this tissue usually produce sex reversal or sterility which is not lethal for the individual. Therefore, gonadal development serves as an excellent model system for investigating questions of cell fate and organogenesis. The studies of human patients showing different types of sex reversal, in combination with the use of transgenic mice and/or gene targeting disruption, have led to the isolation of several genes important for sex development. These include SRY/Sry, encoding the testis-determining factor, Ftz-F1 encoding steroidogenic factor 1 (SF-1) and Wilms' tumor gene (WT-1). However, the mammalian sex differentiation pathway requires the function of a number of additional genes which we are now trying to identify with the help of mRNA differential display technique.

Animals↗
PubMed1994

Human adenovirus encodes two proteins which have opposite effects on accumulation of alternatively spliced mRNAs.

All mRNAs expressed from the adenovirus major late transcription unit have a common, 201-nucleotide-long 5' leader sequence, which consists of three short exons (the tripartite leader). This leader has two variants, either with or without the i-leader exon, which, when present, is spliced between the second and the third exons of the tripartite leader. Previous studies have shown that adenovirus early region 4 (E4) encodes two proteins, E4 open reading frame 3 (E4-ORF3) and E4-ORF6, which are required for efficient expression of mRNAs from the major late transcription unit. These two E4 proteins appear to have redundant activities, and expression of one has been shown to be sufficient for efficient major late mRNA accumulation during a lytic virus infection. In this report, we provide evidence that E4-ORF3 and E4-ORF6 both regulate major late mRNA accumulation by stimulating constitutive splicing. Moreover, we show that the two proteins have different effects on accumulation of alternatively spliced tripartite leader exons. In a DNA transfection assay, E4-ORF3 was shown to facilitate i-leader exon inclusion, while E4-ORF6 preferentially favored i-leader exon skipping. In addition, E4-ORF3 and E4-ORF6 had the same effects on accumulation of alternatively spliced chimeric beta-globin transcripts. This finding suggests that the activities of the two proteins may be of more general relevance and not restricted to splicing of major late tripartite leader-containing pre-mRNAs. Interestingly, E4-ORF6 expression was also shown to stimulate i-leader exon skipping during a lytic virus infection.

Adenoviruses, Human↗
PubMed1993

Two adenovirus proteins with redundant activities in virus growth facilitates tripartite leader mRNA accumulation.

Most adenovirus-specific mRNAs expressed late after infection originate from a single transcription unit, the so-called major late transcription unit. All mRNAs expressed from this unit have in common a 201 nucleotide-long spliced tripartite leader segment attached to their 5' ends. Human adenovirus mutants that carry large deletions in early region 4 (E4) are severely defective in expression of nuclear and cytoplasmic RNA derived from the major late transcription unit. We have previously shown that E4 post-transcriptionally stimulates accumulation of tripartite leader containing mRNAs by a mechanism that requires an intron in the nuclear precursor RNA. To identify the E4 products responsible for this stimulatory effect on tripartite leader mRNA accumulation, we constructed CMV expression vectors encoding single E4 open reading frames (ORFs). By comparing the activity of these plasmids in a transient DNA cotransfection assay we could show that both the E4-ORF3 and E4-ORF6 proteins individually are able to stimulate mRNA accumulation from tripartite leader intron containing transcription units. Furthermore, this stimulatory activity was not dependent on coexpression of other viral gene products. These results are interesting since the same two E4 proteins have been shown to have interchangeable activities in lytic infection, and expression of one has been suggested to be sufficient to substitute for the whole E4 region for virus growth. Finally, we show that the absence of E4 expression during a virus infection results in abnormalities in tripartite leader assembly. This result suggests that E4 proteins may be required for efficient tripartite splicing also during a lytic virus infection.

Adenovirus E1A Proteins↗
PubMed1990

Adenovirus early region 4 stimulates mRNA accumulation via 5' introns.

The adenovirus major late transcription unit accounts for most virus-specific transcription late after infection. All mRNAs expressed from this unit carry a short spliced leader, the so-called tripartite leader, attached to their 5' ends. Here we describe a function for an adenovirus gene product in the control of major late mRNA abundance. We show that early region 4 (E4) stimulates mRNA accumulation from tripartite leader intron-containing transcription units approximately 10-fold in short-term transfection assays. The effect was already detectable in nuclear RNA and was not due to a transcriptional activation through any of the major late promoter elements or through an effect at nuclear to cytoplasmic mRNA transport. A surprising positional effect of the intron was noted. To be E4 responsive, the intron had to be placed close to the pre-mRNA 5' end. The same intron located far downstream in the 3' untranslated region of the mRNA was not E4 responsive. The E4 enhancement was not dependent on specific virus exon or intron sequences. These results suggest that E4 modulates a general pathway in mammalian mRNA formation.

Adenoviruses, Human↗
PubMed1988

Gene product of region E4 of adenovirus type 5 modulates accumulation of certain viral polypeptides.

An adenovirus type 5 mutant, designated H5ilE4I, was constructed in which region E4 was replaced by a cloned cDNA. The cDNA was a copy of an mRNA which exclusively contains open translational reading frames 6 and 7. The phenotype of the mutant was compared with that of the previously characterized E4 mutant H2dl808 and wild-type adenovirus 5. Although the H5ilE4I mutant lacked at least five E4 genes, it was nondefective for growth in HeLa cells. The defects in viral DNA replication, late protein synthesis, and shutoff of host cell protein synthesis associated with the phenotype of the H2dl808 mutant were not observed in HeLa cells infected with the H5ilE4I mutant. However, differences were observed regarding the time of onset of viral DNA replication and the accumulation of the hexon polypeptide as well as the 72-kilodalton adenovirus-specific DNA-binding protein. The results thus indicate that open reading frame 6 or 7 or both contain all genetic information required for viral replication in tissue culture cells, whereas another E4 gene modulates the accumulation of certain viral polypeptides. The early onset of viral DNA replication in H5ilE4I-infected cells may be an indirect effect of the enhanced expression of the 72-kilodalton DNA-binding protein.

Adenoviruses, Human↗
PubMed1987

A novel adenovirus-2 E1A mRNA encoding a protein with transcription activation properties.

Two novel adenovirus-2 early region 1A mRNAs, designated 10S and 11S, have been characterized. They differ from the previously described 9S, 12S and 13S mRNAs by having an additional intron removed during mRNA maturation. The 10S and 11S mRNAs encode proteins with mol. wts of 30 and 35 kd. These proteins are encoded in the same translational reading frame as the 12S and 13S mRNA products and differ by lacking 72 amino acids between position 27 and 98. A functional analysis showed that both the 10S and 11S mRNA products are non-essential for lytic virus growth, and, furthermore, defective in cellular transformation. Interestingly the 11S mRNA product functioned as an efficient transcriptional activator in transient expression assays but was very ineffective as a gene activator during virus growth. Moreover, the virus expressing the 11S cDNA failed to block host cell gene expression although substantial amounts of late proteins were expressed. From the biological properties of the E1A cDNA mutants it was possible to localize two functional domains in the E1A proteins; one region required for transcriptional activation (amino acids 140-185), and a second domain required for adenovirus transformation and the control of viral and cellular gene expression during a lytic infection (amino acids 27-98).

Adenovirus Early Proteins↗