Syntenic mapping and chromosomal localization of bovine α and β interferon genes

The previous assignment of bovine -(IFNA) and -(IFNB) interferon gene families to syntenic group U18 was confirmed with additional cDNA probes and a bovine-rodent hybrid somatic cell panel representing all 29 bovine autosomal syntenic groups. Fluorescent in situ hybridization (FISH) localized these genes to bovine Chromosome (Chr) 8 band 15 and demonstrates that with biotinylated plasmids, as few as five tandemly arrayed sequences can be detected by conventional fluorescent microscopy. This technique can be applied to physical mapping of other multicopy genes in domestic animals.     

Molecular cloning, identification, and chromosomal localization of two MADS box genes in peach （Prunus persica）

MADS box proteins play an important role in floral development. To find genes involved in the floral transition of Prunus species, cDNAs for two MADS box genes, PpMADS1 and PpMADS10, were cloned using degenerate primers and 5'- and 3'- RACE based on the sequence database of P. persica and P. dulcis. The full length of PpMADS1 eDNA is 1, 071bp containing an open reading frame (ORF) of 717bp and coding for a polypeptide of 238 amino acid residues. The full length of PpMADS10 cDNA is 937bp containing an ORF of 633bp and coding for a polypeptide of 210 amino acid residues. Sequence comparison revealed that PpMADS1 and PpMADS10 were highly homologous to genes AP1 and PI in Arabidopsis, respectively. Phylogenetic analysis indicated that PpMADS1 belongs to the euAP1 clade of class A, and PpMADS10 is a member of GLO/PI clade of class B. RT-PCR analysis showed that PpMADS1 was expressed in sepal, petal, carpel, and fruit, which was slightly different from the expression pattern of AP1; PpMADS10 was expressed in petal and stamen, which shared the same expression pattern as PI. Using selective mapping strategy, PpMADS1 was assigned onto the Bin 1:50 on the G1 linkage group between the markers MCO44 and TSA2, and PpMADS10 onto the Bin 1:73 on the same linkage group between the markers Lap-1 and FGA8. Our results provided the basis for further dissection of the two MADS box gene function.     

Polymorphism and chromosomal location of endogenous α-amylase inhibitor genes in common wheat

Summary Polymorphism of an endogenous -amylase inhibitor in wheat was studied using iso-electric focusing followed by monoclonal antibody — based immunoblotting. Ten isoforms of the inhibitor detected in common wheat and its wild counterparts were assigned to five homoeologous loci. Three -amylase inhibitor loci (Isa-1) were identified in common wheat and located on the long arms of chromosomes 2A, 2B and 2D. In a sample of 27 bread wheats, eight durum wheats, and 12 diploid wheat relatives, amphiploids and triticales, a high resolution isoelectric-focusing separation demonstrated two active and one null allele at the Isa-A1, two alleles at the Isa-B1, one allele at the Isa-D1, four alleles at the Isa-S1, and one allele at the Isa-G1 locus. The most frequent electrophoretic pattern of common wheat cultivars consisted of two isoforms, encoded respectively by the Isa-B1b, Isa-D1 a alleles and the Isa-Alnull allele. All the durum wheats had only one inhibitor form controlled by allele Isa-B1b, which was accompanied by the null allele at the Isa-A1 locus.Contribution No. 210 of the Food Science Department, University of Manitoba     

Molecular cloning and chromosomal localization of a human skeletal muscle PP-1γ1 cDNA

Type-1-protein phosphatase (PP-1) activity is reduced in skeletal muscle from human subjects with insulin resistance (Kida et al. 1990). This reduced phosphatase activity probably leads to the abnormal insulin action for glucose storage observed in insulin-resistant subjects. In the present study, a human homolog of rat liver PP-11 cDNA was isolated from human skeletal muscle. The nucleotide sequence contains a 957-nucleotide open reading frame encoding an amino acid sequence identical to that encoded by rat liver PP-11 cDNA. Northern blot analysis shows PP-11-specific mRNA is expressed in human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. PP-11 was localized to human Chromosome 12.     

Cloning and chromosomal localization of a novel gene-encoding a human β2-integrin α subunit

We isolated a partial genomic clone encoding ITGAD, a novel β₂-integrin α subunit. The ITGAD gene is highly homologous to the three previously known α subunit-encoding genes, that compose the β₂ integrin family, in deduced amino acid sequence, intron/exon structure and mapping location (chromosome 16p11).     

Isolation of full-length cDNA and chromosomal localization of human NF-κB modulator NEMO to Xq28

NEMO is an essential component of the IB kinase complex. Others have shown that expression of mouse NEMO can complement the lack of responsiveness to NF-B stimuli in two NEMO-deficient cell lines. Here we report the isolation of a full-length human NEMO cDNA. Virtual translation of human NEMO cDNA predicts a 48-kD coiled-coil protein which shares 87.9% identity and 90.5% similarity with the mouse homolog. By sequence alignment, we mapped the human NEMO gene to chromosome Xq28. We note that the NEMO and the G6PD (glucose-6-phosphate dehydrogenase) loci are arranged in a head-to-head orientation separated by no more than 800 bp. This map location is further supported by the sequence of an alternatively spliced variant of human NEMO mRNA. Thus, human NEMO is an X-linked gene closely adjacent to the G6PD locus.     

Synthesis and anti-leishmanial activity of 1-aryl-β-carboline derivatives against Leishmania donovani

β-carbolines from various natural and synthetic sources have been known to show diverse biological activities. As a part of our current ongoing project to search for potent natural product-derived anti-leishmanial compounds, we have synthesized a series of substituted 1-aryl-β-carboline derivatives. A total of 22 compounds were synthesized and tested in vitro against Leishmania donovani, out of which 6 compounds (4, 5, 10, 11, 19 and 22) showed notably more activity than the standard miltefosine (IC(50) 12.07±0.82 μM), with compound 4 being the most potent (IC(50) 2.16±0.26 μM).     

Molecular characterization of four β-tubulin genes from dinitroaniline susceptible and resistant biotypes of Eleusine indica

Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, resistant biotypes of goosegrass (Eleusine indica) developed in previously susceptible wild-type populations. We have previously reported that -tubulin missense mutations correlate with dinitroaniline response phenotypes (Drp) (Plant Cell 10: 297–308, 1998). In order to ascertain associations of other tubulins with dinitroaniline resistance, four -tubulin cDNA classes (designated TUB1, TUB2, TUB3, and TUB4) were isolated from dinitroaniline-susceptible and -resistant biotypes. Sequence analysis of the four -tubulin cDNA classes identified no missense mutations. Identified nucleotide substitutions did not result in amino acid replacements. These results suggest that the molecular basis of dinitroaniline resistance in goosegrass differs from those of colchicine/dinitroaniline cross-resistant Chlamydomonas reinhardtii and benzimidazole-resistant fungi and yeast. Expression of the four -tubulins was highest in inflorescences. This is in contrast to -tubulin TUA1 that is expressed predominantly in roots. Collectively, these results imply that -tubulin genes are not associated with dinitroaniline resistance in goosegrass. Phylogenetic analysis of the four -tubulins, together with three -tubulins, suggests that the resistant biotype developed independently in multiple locations rather than spreading from one location.     

Cloning,characterisation and expression of the α-tubulin genes of the leech,Hirudo medicinalis

We have isolated two α-tubulin cDNAs from the leech, Hirudo medicinalis. Both encode putative proteins of 451 amino-acids which differ from each other at only two positions. Southern blotting suggests that there are only two α-tubulin genes in the leech. The genes contain two introns and, because of the extremely high homology of the nucleotide sequence from the second intron to the end of the genes, we have inferred that a gene conversion event about 9.5 million years ago has homogenised the Hirudo α-tubulin sequences. Using in situ hybridisation to tissue sections, we have shown that the two genes are probably expressed in all neurons of the leech ganglia and that their spatial distribution remains unchanged during neuronal regeneration. The deduced amino-acid sequences of the leech α-tubulins show that they have greatest similarity to those from a platyhelminth, echiuran and mollusc with rather less to arthropod α-tubulins. The protein sequences of the leech α-tubulins have been compared with representatives of those from across all phyla to determine if any specific feature labels certain isotypes of tubulin for neuronal expression.     

The GCP3-interacting proteins GIP1 and GIP2 are required for γ-tubulin complex protein localization, spindle integrity, and chromosomal stability

Microtubules (MTs) are crucial for both the establishment of cellular polarity and the progression of all mitotic phases leading to karyokinesis and cytokinesis. MT organization and spindle formation rely on the activity of γ-tubulin and associated proteins throughout the cell cycle. To date, the molecular mechanisms modulating γ-tubulin complex location remain largely unknown. In this work, two Arabidopsis thaliana proteins interacting with gamma-tubulin complex protein3 (GCP3), GCP3-interacting protein1 (GIP1) and GIP2, have been characterized. Both GIP genes are ubiquitously expressed in all tissues analyzed. Immunolocalization studies combined with the expression of GIP-green fluorescent protein fusions have shown that GIPs colocalize with γ-tubulin, GCP3, and/or GCP4 and reorganize from the nucleus to the prospindle and the preprophase band in late G2. After nuclear envelope breakdown, they localize on spindle and phragmoplast MTs and on the reforming nuclear envelope of daughter cells. The gip1 gip2 double mutants exhibit severe growth defects and sterility. At the cellular level, they are characterized by MT misorganization and abnormal spindle polarity, resulting in ploidy defects. Altogether, our data show that during mitosis GIPs play a role in γ-tubulin complex localization, spindle stability and chromosomal segregation.