The Acquisition of an Inheritable 50-bp Deletion in the Human mtDNA Control Region Does Not Affect the mtDNA Copy Number in Peripheral Blood Cells

The mitochondrial DNA (mtDNA) control region is believed to play an important biological role in mtDNA replication. Large deletions in this region are rarely found, but when they do occur they might be expected to interfere with the replication of the molecule, thus leading to a reduction of mtDNA copy number. During a survey for mtDNA sequence variations in 5,559 individuals from the general Chinese population and 2,538 individuals with medical disorders, we identified a 50-bp deletion (m.298_347del50) in the mtDNA control region in a member of a healthy Han Chinese family belonging to haplogroup B4c1b2, as suggested by complete mtDNA genome sequencing. This deletion removes the conserved sequence block II (CSBII; region 299-315) and the replication primer location (region 317-321). However, quantification of the mtDNA copy number in this subject showed a value within a range that was observed in 20 healthy subjects without the deletion. The deletion was detected in the hair samples of the maternal relatives of the subject and exhibited variable heteroplasmy. Our current observation, together with a recent report for a benign 154-bp deletion in the mtDNA control region, suggests that the control of mtDNA replication may be more complex than we had thought. Hum Mutat 31:538-543, 2010. (C) 2010 Wiley-Liss, Inc.

Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites

With current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10(-4) to 10(-5)) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering.

Rapid and parallel chromosomal number reductions in muntjac deer inferred from mitochondrial DNA phylogeny

Muntjac deer (Muntiacinae, Cervidae) are of great interest in evolutionary studies because of their dramatic chromosome variations and recent discoveries of several new species. In this paper, we analyze the evolution of karyotypes of muntjac deer in the context of a phylogeny which is based on 1,844-bp mitochondrial DNA sequences of seven generally recognized species in the muntjac subfamily. The phylogenetic results support the hypothesis that karyotypic evolution in muntjac deer has proceeded via reduction in diploid number. However, the reduction in number is not always linear, i.e., not strictly following the order: 46-->14/13-->8/9-->6/7. For example, Muntiacus muntjak (2n = 6/7) shares a common ancestor with Muntiacus feae (2n = 13/14), which indicates that its karyotype was derived in parallel with M. feae's from an ancestral karyotype of 2n greater than or equal to 13/14. The newly discovered giant muntjac (Muntiacus vuquangensis) may represent another pa;allel reduction lineage from the ancestral 2n = 46 karyotype. Our phylogenetic results indicate that the giant muntjac is relatively closer to Muntiacus reevesi than to other muntjacs and may be placed in the genus Muntiacus. Analyses of sequence divergence reveal that the rate of change in chromosome number in muntjac deer is one of the fastest in vertebrates. Within the muntjac subfamily, the fastest evolutionary rate is found in the Fea's lineage, in which two species with different karyotypes diverged in around 0.5 Myr.

Temporal and spatial variations in the species composition, distribution, and abundance of protozoa in East Dongting lake, China

Investigations of protozoa were carried out during four surveys of East Dongting Lake, China. A total of 160 protozoan species belonging to 71 genera was identified, of which 53 were flagellates, 37 sarcodines, and 70 ciliates. Among them, Peritrichida (32.6% of frequency), Arcellinida (16.2%), Volvocales (13.61/6), Peridiniales (13.1%), and Chrysomonadales (9.1%) were the main groups and contributed to 84.5% of the overall species. Ciliates were mainly composed of sessile species and small species. The total protozoan abundance varied from 2,400 cells L-1 to 20,250 cells L-1. The highest protozoan abundance occurred in spring; the lowest number was in autumn. The highest abundance of ciliates occurred in spring and winter, whereas flagellates developed the highest abundance in,summer and autumn. Pearson correlation analysis and linear regressions indicated that chlorophyll a and water velocity were the main factors affecting ternporal and spatial variations of the protozoan abundance.