Transposition of Tnr1 in rice genomes to 5′-PuTAPy-3′ sites,duplicating the TA sequence

Tnr1 is a repetitive sequence in rice with several features characteristic of a transposable DNA element. Its copy number was estimated to be about 3500 per haploid genome by slot-blot hybridization. We have isolated six members of Tnr1 located at different loci by PCR (polymerase chain reaction) and determined their nucleotide sequences. The Tnr1 elements were similar in size and highly homologous (about 85%) to the Tnr1 sequence identified first in the Waxy gene in Oryza glaberrima. A consensus sequence of 235 by could be derived from the nucleotide sequences of all the Tnr1 members. The consensus sequence showed that base substitutions occurred frequently in Tnr1 by transition, and that Tnr1 has terminal inverted repeat sequences of 75 bp. Almost all the chromosomal sequences that flank the Tnr1 members were 5′-PuTA-3′ and 5′-TAPy-3′, indicating that Tnr1 transposed to 5′-PuTAPy-3′ sites, duplicating the TA sequence. PCR-amplified fragments from some rice species did not contain the Tnr1 members at corresponding loci. Comparison of nucleotide sequences of the fragments with or without a Tnr1 member confirmed preferential transposition of Tnr1 to 5′-PuTAPy-3′ sites, duplicating the TA sequence. One amplified sequence suggested that imprecise excision had occurred to remove a DNA segment containing a Tnr1 member and its neighboring sequences at the Waxy locus of rice species with genome types other than AA. We also present data that may suggest that Tnr1 is a defective form of an autonomous transposable element.     

Hepatocyte nuclear factor 4α regulates the expression of the murine pyruvate carboxylase gene through the HNF4-specific binding motif in its proximal promoter

Pyruvate carboxylase (PC) is the first regulatory enzyme of gluconeogenesis. Here we report that the proximal promoter of the murine PC gene contains three binding sites for hepatocyte nuclear factor 4α (HNF4α). These sites include the classical direct repeat 1 (DR1) (− 386/− 374), non-perfect DR1 (− 118/− 106) and HNF4α-specific binding motif (H4-SBM) (− 26/− 14). Under basal conditions, mutation of the non-perfect DR1 decreased promoter activity by 50%, whereas mutation of neither the DR1 nor the H4-SBM had any effect. In marked contrast, only mutation of the H4-SBM decreased HNF4α-transactivation of the promoter activity by 65%. EMSA revealed that HNF4α binds to the DR1site and H4-SBM with similar affinity while it binds poorly to the non-perfect DR1. Interestingly, this non-perfect DR1 also coincides with two E-boxes. Mutation of the non-perfect DR1 together with the nearby E-box reduced USF1- but not USF2-transactivation of promoter activity, suggesting that USF1 partly contributes to the basal activity of the promoter. Substitution of the H4-SBM with the DR1 marginally reduced the basal promoter activity but did not eliminate HNF4α-transactivation, suggesting that HNF4α can exert its effect via DR1 within this promoter context. ChIP-assay confirmed that HNF4α is associated with the H4-SBM. Suppression of HNF4α expression in AML12 cells down-regulated PC mRNA and PC protein by 60% and 50%, respectively, confirming that PC is a target of HNF4α. We also propose a model for differential regulation of P1 promoter of PC gene in adipose tissue and liver.     

Identification of β 1 → 4 glucan chains as part of a fraction of slime synthesized within the dictyosomes of maize root caps

Summary The protective polysaccharides synthesized by the outer root-cap cells of maize have been prepared in radioactive and non-radioactive form and studied using the techniques oftrans-elimination, gel filtration and partial hydrolysis under acid and alkaline conditions. The results indicate that the slime consists of a central 1 4 linked glucan rendered soluble by a coating of hydrophilic polysaccharides linked both covalently and non-covalently. The covalently-linked polysaccharide is relatively rich in galacturonic acid and fucose in regions near the central glucan. It is likely that the synthesis of the slime, including the glucan component, takes place within the dictyosome sacs and vesicles and this has important consequences for ideas on the sites of 1 4 glucan synthesis within plant cells.     

Identification and sequence analysis of the keratin-associated protein 24‐1 (KAP24-1) gene homologue in sheep

Keratin-associated proteins (KAPs) are major structural components of hair and wool fibres, and play a critical role in determining the properties of the fibre. While over 100 KAP genes that have been grouped into 27 KAP families have been identified in mammals, most homologues remain unidentified in sheep.     

Expression of the human granulocyte–macrophage colony stimulating factor (hGM-CSF) gene under control of the 5′-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice

Expression of the human granulocyte–macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5′-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.9 to 14 μg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7 μg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected “protective or enhancer effect” from the MAR element on the hGM-CSF gene expression was not observed.     

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.     

Sequencing of the human vascular endothelial growth factor (VEGF) 3′ untranslated region (UTR): conservation of five hypoxia-inducible RNA-protein binding sites

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor whose mRNA expression is induced by hypoxia. This induction is due in large part to an increase in the stability of its mRNA. The RNA sequences and cognate proteins responsible for this increased stability with hypoxia are not well understood. In order to identify regions of functional importance in the 3′UTR of VEGF mRNA, we have sequenced the human VEGF 3′UTR and compared it to the rat sequence. Overall sequence homology was 82% with complete conservation of all four potential polyadenylation signals and both nonameric instability elements. Five hypoxia-inducible RNA protein-binding (HI-RPB) sites were identified by RNA electromobility shift assay (EMSA) in the human and rat genes. EMSA and competition studies suggest that these sites bind a similar or related protein complex. On average, the five sites were 95% conserved at the nucleotide level between the rat and corresponding human sequence. This conservation taken together with several previously described, independent correlations between the presence of these RNA-protein complexes and an increase in VEGF mRNA stability suggest an important functional role for these sites in mediating hypoxia-inducible VEGF mRNA stability.