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Biomedical subjects

L Montoliu

Publications and source records attributed to L Montoliu.

29 records · Page 2Linked to original sources
PubMed1994

Germ line transmission of yeast artificial chromosomes in transgenic mice.

Several groups have recently reported the successful generation of transgenic mice harbouring yeast artificial chromosomes (YACs). Different methodological approaches have been shown to produce similar results, namely, the faithful expression of the transgenes carried on YAC DNA. In this paper, we compare the reported techniques for obtaining transgenic mice carrying YACs using a 250-kb YAC bearing the mouse tyrosinase gene. These methods include: microinjection of gel-purified YAC DNA into pronuclei of fertilized mouse oocytes, yeast spheroblast fusion with embryonic stem (ES) cells and lipofection of YAC DNA into ES cells. Taken together, these reports show that the delivery of large genomic regions covering a gene of interest (such as those cloned in YAC vectors) is feasible, and will ensure appropriate temporal and spatial expression of the transgene at a level comparable to that of the endogenous counterpart.

Animals↗
PubMed1994

Analysis of the cAMP response on liver-specific gene expression in transgenic mice.

Expression of many genes is modulated by intracellular variations of cyclic AMP (cAMP) levels in response to different signals from the environment. This regulation is mediated via a cAMP-response element (CRE). This report addresses the role of cAMP in the physiological activation of a subset of liver-specific genes which are perinatally activated. The tyrosine aminotransferase (TAT) gene and other genes such as phosphoenolpyruvate carboxyquinase (PEPCK) and glucose-6-phosphatase, involved in gluconeogenesis, belong to this category. CRE elements derived from the rat TAT-3.6 kb enhancer have been positioned in chimeric constructs, such that the activity of the reporter gene LacZ is dependent on cAMP. The tissue-specificity of these constructs is guaranteed by the presence of the liver-specific enhancers of the alpha fetoprotein gene. These constructs have been tested in cells and transgenic mice demonstrating cAMP regulation, liver-specific expression and perinatal activation of the reporter gene. The CRE is recognized by a number of related proteins of which the cAMP-response element-binding factor (CREB) has been best studied. To assess the role of CREB in the in vivo transduction of cAMP signalling, mice deficient in CREB protein have been generated by homologous recombination in embryonic stem (ES) cells. Homozygous mutant mice, although recovering at a lower ratio than expected, do not display impairment of growth or development. The cAMP-dependent LacZ transgenic mice in a CREB mutant genetic background also show perinatal activation of the reporter gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

A method for the generation of YAC transgenic mice by pronuclear microinjection.

Yeast artificial chromosomes (YACs) represent the latest generation of vectors which have the great advantage of large insert size. The introduction of YACs into mammalian cells and organisms has become an important goal, since it offers the potential to study the control of large and complex transcription units and identify genes by complementation. Microinjection into the nucleus is the most direct and efficient way of delivering YAC DNA into cells, but requires the purification of the YAC from the remaining yeast chromosomes. Here we describe a detailed method for the isolation of pure, intact and highly concentrated YAC DNA. As a model system the murine tyrosinase gene was chosen and four YACs covering this locus were isolated. Introduction by homologous recombination in yeast of sequences permitting YAC amplification greatly facilitated the isolation of YAC DNA at high concentrations. YAC DNA stabilized in a salt and polyamine containing buffer did not compromise the survival of microinjected oocytes and was suitable for the generation of transgenic mice. Applications and benefits of this technique will be discussed.

Animals↗
PubMed1993

A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice.

Expression of transgenes in mice often fails to follow the normal temporal and spatial pattern and to reach the same level as the endogenous copies. Only in exceptional cases has position-independent and copy number-dependent expression been reproduced. The size constraint of standard constructs may prevent the inclusion of important remote regulatory elements. Yeast artificial chromosomes (YACs) provide a means of cloning large DNA fragments and the transfer of YAC DNA into somatic cells has been reported. We have previously produced transgenic mice carrying a 35 kilobase YAC construct. Here we report the transfer of a 250 kilobase YAC covering the mouse tyrosinase gene into mice by pronuclear injection of gel-purified YAC DNA. The YAC was inserted into the mouse genome without major rearrangements and expression of the YAC-borne tyrosinase gene resulted in complete rescue of the albino phenotype of the recipient mice. Expression from the transgene reached levels comparable to that of the endogenous gene and showed copy number dependence and position independence.

Animals↗
PubMed1992

Transgenic mice generated by pronuclear injection of a yeast artificial chromosome.

Transgenic mice have become invaluable for analysing gene function and regulation in vivo. However, the size of constructs injected has been limited by the cloning capacity of conventional vectors, a constraint that could be overcome with yeast artificial chromosomes (YACs). We investigated the feasibility of making transgenic mice with YACs by pronuclear injection of a small YAC carrying a gene encoding tyrosinase. Use of a vector with a conditional centromere allowed fifteenfold amplification of the YAC in yeast and its recovery in high yield. The albino phenotype of the recipient mice was rescued demonstrating the correct expression of the tyrosine gene from the construct. Furthermore, the telomeric sequences added by the yeast integrated into the mouse genome and did not reduce efficiency of integration. Using this technique future experiments with longer YACs will allow the expression of gene complexes such as Hox and the globin gene clusters to be analysed in transgenic animals.

Animals↗
PubMed1992

Structure and expression of the lignin O-methyltransferase gene from Zea mays L.

The isolation and characterization of cDNA and homologous genomic clones encoding the lignin O-methyltransferase (OMT) from maize is reported. The cDNA clone has been isolated by differential screening of maize root cDNA library. Southern analysis indicates that a single gene codes for this protein. The genomic sequence contains a single 916 bp intron. The deduced protein sequence from DNA shares significant homology with the recently reported lignin-bispecific caffeic acid/5-hydroxyferulic OMTs from alfalfa and aspen. It also shares homology with OMTs from bovine pineal glands and a purple non-sulfur photosynthetic bacterium. The mRNA of this gene is present at different levels in distinct organs of the plant with the highest accumulation detected in the elongation zone of roots. Bacterial extracts from clones containing the maize OMT cDNA show an activity in methylation of caffeic acid to ferulic acid comparable to that existing in the plant extracts. These results indicate that the described gene encodes the caffeic acid 3-O-methyltransferase (COMT) involved in the lignin bio-synthesis of maize.

Amino Acid Sequence↗
PubMed1990

Multiple polyadenylation sites are active in the alpha 1-tubulin gene from Zea mays.

Two cDNA clones (MR2 and MR29) encoding the same alpha-tubulin isotype (alpha 1) have been identified and characterized from maize roots. The sequence of these two cDNA clones is identical to a previously described cDNA clone (MR19) except in the location of their polyadenylation site. The sequence of the cDNA and its homologous genomic clone (MG19/14) shows two putative polyadenylation signals which could direct the variable 3' processing of the observed transcripts. Endonuclease S1 protection analysis in this 3' flanking region confirms the presence in the alpha 1-tubulin gene from Zea mays of these two main functional polyadenylation sites and possibly other related ones. The relative accumulation of RNAs bearing the two main polyadenylation sites has been tested by using a RNA-slot analysis of several tissues of the plant. It appears that a higher proportion of shorter mRNA species is found in actively dividing tissues.

Base Sequence↗
PubMed1990

The Tub alpha 3 gene from Zea mays: structure and expression in dividing plant tissues.

A gene (Tub alpha 3) coding for an alpha-Tub, expressed in dividing tissues, has been cloned from Zea mays. The deduced amino acid (aa) sequence, 450 aa long, is very similar to the other plant alpha-Tub (85-89% homology) so far reported, and in particular to the other two aa sequences (alpha 1-Tub and alpha 2-Tub) already published from the same species (93% homology). The genomic structure is also very similar, having three introns located at the same positions as in the Tub alpha 1 and Tub alpha 2 genes, one of them placed at the same position in the homologous genes from Arabidopsis thaliana. Nevertheless, the noncoding sequences are very different from the two other maize genomic sequences. In particular, no homology has been found either in the 5' upstream or in the 3'-untranslated sequences. Using specific 3' probes, it has been possible to detect the mRNA coded by this gene in many of the plant organs measured, but its highest abundance is observed in the organs rich in dividing cells, a pattern correlated with that of the histone H4-encoding gene. A cDNA clone has been identified in maize coleoptiles and sequenced, confirming the expression of the Tub alpha 3 in this organ. No preferential accumulation in any organ of the plant was found, in contrast with what was observed in the Tub alpha 1 and Tub alpha 2 genes already described. The Tub alpha gene family seems to consist in maize by at least two groups of homologous sequences, each one including a maximum of two or three coding units.

Amino Acid Sequence↗
PubMed1990

A tandem of alpha-tubulin genes preferentially expressed in radicular tissues from Zea mays.

The identification of a cDNA (MR19) corresponding to a maize alpha-tubulin and homologous genomic clones (MG19/6 and MG19/14) is described. The cDNA has been isolated by differential screening of a cDNA maize root library. We have found two alpha-tubulin genes in a tandem arrangement in the genomic clones, separated by approximately 1.5 kbp. One of the genes (gene I) contains an identical nucleotide sequence which corresponds to the cDNA clone. The two deduced proteins from DNA sequences are very similar (only two conservative replacements in 451 amino acids) and they share a high homology as compared with the published alpha-tubulin sequences from other systems and in particular with the Arabidopsis thaliana and Chlamydomonas reinhardtii sequences reported. The structure of both genes is also very similar; it includes two introns, of 1.7 kbp and 0.8 kbp respectively, in each gene and only one intron placed at a homologous position in relation to Arabidopsis thaliana genes. By using specific 3' probes it appears that both genes are preferentially expressed in the radicular system of the plant. The alpha-tubulin gene family of Zea mays seems to be represented by at least 3 or 4 members.

Amino Acid Sequence↗
PubMed1989

Sequence of rat skeletal muscle phosphoglycerate mutase cDNA.

A cDNA clone coding rat skeletal muscle phosphoglycerate mutase was isolated from a rat muscle lambda gt10 cDNA library and its sequence was determined. The deduced protein possesses 252 amino acids and is 94% homologous with respect to human muscle phosphoglycerate mutase. No amino acids changes occur at the active site and structural predictions suggest strong conformational homologies with other enzymes of the mutase family.

Amino Acid Sequence↗