Biological function of a tobacco homeobox gene, NTH15, was studied by producing two different types of transgenic tobacco, one of which was transformed with NTH15::GUS and the other was transformed with CaMV 35S promoter::antisense NTH15. The GUS staining around shoot apical meristem (SAM) in transgenic plants with NTH15::GUS revealed that the NTH15 expression is specifically localized in the dorsal region of young leaf primordia. The transgenic plants transformed with the antisense construct lost the expression of the endogenous transcript around SAM and also showed alterations in leaf morphology. These transformants formed leaves with disorganized pinnate venation. Midribs of the malformed leaves also lost crescent-shaped vascular systems by ectopic cell vacuolization in their adaxial side. These observations suggest suppression of the NTH15 expression around SAM causes loss of dorsoventrality in midribs. Taken together with the observations of the NTH15 expression pattern, it may be important for the dorsoventral development of leaves that the NTH15 gene is expressed in the adaxial side of young leaf primordia.
The cDNA encoding the Drosophila ananassae ornithine aminotransferase (OAT, EC 220.127.116.11) precursor has been cloned and characterized. The predicted OAT protein sequence is 433 amino acids long with a molecular mass of 47,352 Da and is highly homologous to a mammalian OAT, which is a mitochondrial matrix enzyme and is matured by processing of its amino terminal presequence peptide. The Drosophila OAT has characteristics of leader peptides present in mitochondrial proteins. Immunoblotting experiments using polyclonal antibodies against the partial sequence of the OAT protein revealed that the OAT monomer has a molecular mass of 44 kDa. These results suggest that the Drosophila OAT is also processed and localized in the mitochondria. Quantitation of the OAT mRNA and measurement of the OAT activity during fly development show that OAT is expressed at high levels in the fat body of the third instar larvae in both D. ananassae and D. melanogaster.
Genetic variation in total mRNA level of the six hsp genes (hsp22, hsp23, hsp26, hsp27, hsp70 and hsp82) in the presence of heat shock was investigated by using seventy-four second- and seventy third-chromosome lines of Drosophila melanogaster which have the same genetic background derived from a highly inbred stock. There was significant variation in all the six hsp genes for both the second- and third-chromosome lines except for the hsp22 gene of the third-chromosome lines. Although all the structural genes of the heat shock proteins are localized on the third chromosome, the estimates of genetic variance for the second-chromosome lines were larger than those for the third-chromosome lines. Highly significant correlations between total mRNA level of the different hsp genes in all the combinations of the six hsp genes using the second-chromosome lines were found, but some of correlations for the third-chromosome lines were not significant. These results suggest that some second chromosome variants have similar effects on the expression of the different genes.
The phylogenetic relationship among the salmonid fishes of the genus Oncorhynchus has been analyzed using various kinds of markers for a long time. However, there are three major disagreements among those studies; (1) the authenticity of the Pacific salmon group as a monophyletic cluster, (2) the phylogenetic relationship among three Pacific salmons (pink salmon, sockeye salmon, and chum salmon), and (3) the phylogenetic position of masu salmon. We used allozyme electrophoresis to clarify the phylogenetic relationship between the Pacific salmon group and the Pacific trout group. Furthermore, we reanalysed published mitochondrial DNA D-loop sequences (Shedlock et al., 1992). Allozymic data and mtDNA data indicated the following consistent results; (1) all Pacific salmons formed a monophyletic cluster, (2) chum salmon and pink salmon were clustered within those Pacific salmons, (3) masu salmon formed a cluster with other Pacific salmons and diverged first in this group.
Improved imaging procedures were developed and employed for the construction of a quantitative chromosome map based on condensation pattern. The condensa-tion pattern is uneven condensation along a chromosomal axis resulting from differential condensation of chromatin fibers at prometaphase appearing commonly in small plant chromosomes, represented by the genus Brassica. Brassica chromosomes have distinct condensation patterns at the prometaphase stage. The procedures for quantitative mapping are divided into six parts: (i) capture of images, (ii) adjustment of light distortion, (iii) measurements of an image parameter, CP, or density profiles and length of chromosomal arms, (iv) gray value adjustments between homologous chromosomes, (v) adjustment of chromosomal lengths and obtaining the standard CP, or graygram, and (vi) thresholding graygram for a quantitative chromosome map or an idiogram. The quantitative chromosome map shows well the characteristics of the Brassica chromosomes. The series of imaging procedures enable construction of quantitative chromosome maps and can be employed for a wide range of species having various chromosome numbers. The quantitative chromosome map constructed by the improved imaging currently developed is an aid to cytogenetical and genome analysis of plant species.
The orientation of RFLP linkage map and the assignment of RFLP markers to the restricted chromosomal segment were determined on seven pachytene chromosomes (1, 3, 4, 5, 8, 9 and 12) of rice. A total of 24 interchange breakpoints were detected by pachytene analysis of reciprocal translocation (RT) heterozygotes derived from crosses between 12 RT lines and normal diploid varieties. Further, 19 strains of japonica-indica hybrid tertiary trisomics were developed and provided for RFLP gene dosage analysis, resulting in locating 32 breakpoints of the extra tertiary chromosomes within genetic linkage segments defined by RFLP markers. From these results, the orientation of RFLP linkage groups on pachytene chromosomes and the assignment of 113 RFLP markers and four cloned rice genes to chromosome arms were conducted, together with the delimitation of centromeres on 6 RFLP linkage groups (1, 3, 4, 8, 9 and 12). Meanwhile, 15 groups of RFLP markers were incorporated with the restricted segments of pachytene chromosome. Compared with RFLP map, the physical distance of a part of the long arm of chromosome 1 was 5.4 to 8.8 cM/μm at the pachytene stage.
Southern blotting of HindIII-digested genomic DNAs from the female and male Oriental white stork (Ciconia boyciana) with the EE0.6 probe cloned from the W chromosome of chicken (Gallus domesticus) produced a 3.0-kb W chromosome-derived band specific to the female and a 3.5-kb Z chromosome-derived band common to both sexes. These two genomic fragments were cloned and the counter-part sequences to that of EE0.6 in these fragments, XH0.6 and XH0.6RSM (XH0.6-related sequence in the male), respectively, were subcloned. Nucleotide sequences of XH0.6 and XH0.6RSM showed 92% identity. PCR using a set of primer sequences from XH0.6, which differed several nucleotides from those in XH0.6RSM, amplified an about 300-bp genomic sequence only from the female C. boyciana. This method was applied successfully to identify the sex of individual young birds of C. boyciana, an endangered special natural monument in Japan and whose sexes are unidentifiable from their external morphology.