Primate evolution of a human chromosome 1 hypervariable repetitive element

Summary The clone designated hMF #1 represents a clustered DNA family, located on chromosome 1, consisting of tandem arrays displaying a monomeric length of 40 bp and a repetition frequency of approximately 7×10³ copies per haploid genome. The sequence hMF #1 reveals multiple restriction fragment length polymorphisms (RFLPs) when human genomic DNA is digested with a variety of 4–6-bp recognition sequence restriction enzymes (i.e., Taq I, Eco RI, Pst I, etc.). When hamster and mouse genomic DNA was digested and analyzed, no cross-species homology could be observed. Further investigation revealed considerable hybridization in the higher primates (chimpanzee, gorilla, and orangutan) as well as some monkey species.The evolutionary relationship of this repetitive DNA sequence, found in humans, to that of other primates was explored using two hybridization methods: DNA dot blot to establish copy number and Southern DNA analysis to examine the complexity of the RFLPs. Homology to the hMF #1 sequence was found throughout the suborder Anthropoidea in 14 ape and New and Old World monkey species. However the sequence was absent in one species of the suborder Prosimii. Several discrepancies between established evolutionary relationships and those predicted by hMF #1 exist, which suggests that repetitive elements of this type are not reliable indicators of phylogenetic branching patterns. The phenomenon of marked diversity between sequence homologies and copy numbers of dispersed repetitive DNA of closely related species has been observed inDrosophila mice,Galago, and higher primates. We report here a similar phenomenon for a clustered repeat that may have originated at an early stage of primate evolution.     

Genotype-specific response of cultured broccoli (Brassica oleracea var. italica) anthers to cytokinins

Anther culture medium was prepared with different types and concentrations of cytokinins to gain greater insight into the control of embryo formation during Brassica oleracea L. var. italica (broccoli) anther culture. The independent addition of the four cytokinins tested had widely divergent effects dependent upon cytokinin concentration and the genetic background of the test plants. All cytokinins were generally inhibitory at high concentrations, however, individual plants showed significant stimulation of embyro formation at typical physiological levels. The influence of cytokinins was highly cultivar-specific, some lines were stimulated, others inhibited and still other test lines were largely unaffected. Although the addition of cytokinins was needed for embryo formation for some plants, in no instance were cytokinins able to replace the inductive effect of high-temperature treatments.     

A Gly1127Ser mutation in an EGF-like domain of the fibrillin-1 gene is a risk factor for ascending aortic aneurysm and dissection.

Ascending aortic disease, ranging from mild aortic root enlargement to aneurysm and/or dissection, has been identified in 10 individuals of a kindred, none of whom had classical Marfan syndrome (MFS). Single-strand conformation analysis of the entire fibrillin-1 (FBN1) cDNA of an affected family member revealed a G-to-A transition at nucleotide 3379, predicting a Gly1127Ser substitution. The glycine in this position is highly conserved in EGF-like domains of FBN1 and other proteins. This mutation was present in 9 of 10 affected family members and in 1 young unaffected member but was not found in other unaffected members, in 168 chromosomes from normal controls, and in 188 chromosomes from other individuals with MFS or related phenotypes. FBN1 intragenic marker haplotypes ruled out the possibility that the other allele played a significant role in modulating the phenotype in this family. Pulse-chase studies revealed normal fibrillin synthesis but reduced fibrillin deposition into the extracellular matrix in cultured fibroblasts from a Gly1127Ser carrier. We postulate that the Gly1127Ser FBN1 mutation is responsible for reduced matrix deposition. We suggest that mutations such as this one may disrupt EGF-like domain folding less drastically than do substitutions of cysteine or of other amino acids important for calcium-binding that cause classical MFS. The Gly1127Ser mutation, therefore, produces a mild form of autosomal dominantly inherited weakness of elastic tissue, which predisposes to ascending aortic aneurysm and dissection later in life.     

On the multiplicity of platelet prostaglandin receptors: II. The use of N-0164 for distinguishing the loct of action for PGI2, PGD2, PGE2 and hydantoin analogs

The ω-chain variant analogs of prostacyclin (PGI₂) and PGD₂ in which the n-amyl side-chain has been replaced by a cyclohexyl group have been prepared and their cardiovascular activities have been compared to those of BW-245C(Fig. 1)(1) a potent anti-aggregatory vasodilator bearing a cyclohexyl-terminated side-chain on a hydantoin skeleton. The cyclohexyl group has little effect on PGI₂, but converts PGD₂ to a long lasting hypotensive agent and increases the platelet anti-aggregatory potency of PGD₂ by a factor of 8. The prostaglandin antagonist N-0164 selectively blocks the anti-aggregatory actions of PGD₂, cyclohexyl-PGD₂, and BW-245C; with essentially no effect on PGI₂, cyclohexyl-PGI₂ and PGE₂ at comparably effective doses. The latter observation is contrary to an earlier report by MacIntyre (2,3), but supports the view that the anti-aggregatory effect of high doses of PGE₂ ($\text{EC}{}_{50}\text{=50μ}\text{M}$) is mediated by the PGI₂ receptor (4). The hydantoin acts at the platelet PGD₂ receptor.     

Genetic stability of the D1Z2 region: implications for DNA genotyping and paternity testing.

The aim of the present study was to examine a single locus variable number tandem repeat for the purpose of DNA genotyping ("fingerprinting"). DNAs of 175 individuals from five ethnic groups (Black, Chinese, Japanese, Caucasian, and Melanesian) were analyzed. Restriction fragment length polymorphic analysis of random individuals revealed individual specific DNA patterns in all but one group. Among 20 Melanesian inhabitants of the Vanuatu islands in the southwest Pacific, three individuals were found to share a common pattern. This island population represents a "genetic isolate" and illustrates the importance of carrying out population studies on individual ethnic groups of interest. The complexity and the genetic stability of the D1Z2 region as revealed by the probe hMF1 make it an excellent candidate for DNA genotyping in paternity testing as 101 Caucasian individuals each had unique patterns for PstI and SinI digests.     

Specimen housing unit for cinemicrographic studies in the vertical plane.

The destructive action of chlorine on the pathogenic Naegleria fowleri and Acanthamoeba culbertsoni, the nonpathogenic N. gruberi, and an avirulent Acanthamoeba isolate was investigated. N fowleri is somewhat more sensitive to chlorine than N. gruberi, whereas the two Acanthamoeba strains are very resistant. This study yields information needed for the destruction of amoebic cysts in drinking water and swimming pools. It also gives some explanation for the occurence of Acanthamoeba strains in these waters.     

High-Throughput Massively Parallel Sequencing for Fetal Aneuploidy Detection from Maternal Plasma

Background

Circulating cell-free (ccf) fetal DNA comprises 3–20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance.

Methods

Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13.

Results

Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively.

Conclusions

These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.     

Conomarphins cause paralysis in mollusk: Critical and tunable structural elements for bioactivity

Two conomarphins were purified as the major component of the venom of Conus eburneus. Conomarphins Eb1 and Eb2 showed biological activity in the mollusk Pomacea padulosa, causing sluggishness and retraction of siphon, foot, and cephalic tentacles. To further probe the effects of conserved amino acids and posttranslational modifications in conomarphins, we prepared four synthetic analogues: conomarphin Eb1 Hyp10Pro, Hyp10Ala, d ‐Phe13Ala, and l ‐Phe13 variants. Structure‐activity relationship analysis indicated that d ‐Phe13 is critical to the biological activity of conomarphins. In contrast, amino acid changes at position 10 and removal of posttranslational modification in Hyp10Pro can be tolerated. The high expression level and observed mollusk activity of conomarphins may suggest their potential role as defensive arsenal of Conoidean snails against other predatory gastropods.