Gene mapping on maize pachytene chromosomes by in situ hybridization

A sensitive in situ hybridization technique which was effective for mapping genes of low copy number on human metaphase chromosomes was used for gene mapping on maize pachytene chromosomes. A cloned genomic EcoR1 fragment of 10.8 kb, containing most or all of the sequence encoding the Waxy locus mRNA, was used as the probe. Southern DNA blotting analyses performed by Shure et al. (1983) indicated that the Waxy locus was a single copy sequence. In our in situ hybridization experiment, the probe hybridized to a specific site on chromosome 9. Labeling at this site was detected in 48.6% of 154 randomly selected copies of chromosome 9. To test the sensitivity of the method, subclones of the fragment with insert sizes of 6.6, 4.7, 3.5, 2.3, 1.9 and 0.8 kb were used for in situ hybridizations. Labeling efficiency for each probe was determined. The data showed that a single copy probe of 1.9 kb could be detected at the correct position in 18% of 183 randomly selected number 9 chromosomes.     

The mop1 (mediator of paramutation1) mutant progressively reactivates one of the two genes encoded by the MuDR transposon in maize

Transposons make up a sizable portion of most genomes, and most organisms have evolved mechanisms to silence them. In maize, silencing of the Mutator family of transposons is associated with methylation of the terminal inverted repeats (TIRs) surrounding the autonomous element and loss of mudrA expression (the transposase) as well as mudrB (a gene involved in insertional activity). We have previously reported that a mutation that suppresses paramutation in maize, mop1, also hypomethylates Mu1 elements and restores somatic activity to silenced MuDR elements. Here, we describe the progressive reactivation of silenced mudrA after several generations in a mop1 background. In mop1 mutants, the TIRA becomes hypomethylated immediately, but mudrA expression and significant somatic reactivation is not observed until silenced MuDR has been exposed to mop1 for several generations. In subsequent generations, individuals that are heterozygous or wild type for the Mop1 allele continue to exhibit hypomethylation at Mu1 and mudrA TIRs as well as somatic activity and high levels of mudrA expression. Thus, mudrA silencing can be progressively and heritably reversed. Conversely, mudrB expression is never restored, its TIR remains methylated, and new insertions of Mu elements are not observed. These data suggest that mudrA and mudrB silencing may be maintained via distinct mechanisms.     

玉米近缘种中玉米着丝粒序列的FISH检测

为分析玉米着丝粒卫星DNA(CentC)和着丝粒反转录转座子(CRM)在玉米种的亚种及近缘种中的保守性,采用双色荧光原位杂交检测了这两种重复序列在墨西哥玉米、二倍体多年生类玉米、多年生类玉米、摩擦禾、薏苡、高粱中的存在和分布。CentC、CRM探针在墨西哥玉米、二倍体多年生类玉米和多年生类玉米的所有染色体的着丝粒区产生了强或较强的杂交信号, 而且不同染色体的杂交信号的强度存在差异, 表明两种玉米着丝粒重复序列在不同着丝粒中的数量不同; 此外, 部分着丝粒中的CentC和CRM信号的强度存在差异, 不完全重叠。CentC、CRM探针仅在摩擦禾的多数染色体的着丝粒区产生了弱的杂交信号。在薏苡和高粱中仅测检到主要分布在着丝粒区的较强或强的CRM信号。这些结果表明, CentC在玉米种的亚种间及玉蜀黍属的物种间高度保守, 在与玉蜀黍属亲缘关系最近的摩擦禾属物种中也具有较高的保守性; CRM在与玉蜀黍属亲缘关系较近和较远的禾本科种属中都具有保守性。     

Expression of collagen type transcripts in chick embryonic bone detected by in situ cDNA-mRNA hybridization

The development of the chick embryonic calvarium, an intramembranous bone, is characterized by direct differentiation of cranial ectomesenchymal cells into osteoblasts without the formation of a cartilage anlage. Collagen biosynthesis remains predominantly as type I in the calvaria. However, in severely calcium-deficient chick embryos maintained in shell-less (SL) culture, cartilage-specific type II collagen is synthesized by the calvaria. Immunohistochemistry localized the cells expressing type II collagen to undermineralized regions of the SL bone. In this study, collagen gene expression in bones of normal (N) and calcium-deficient SL chick embryos was examined at Incubation Day 14 by in situ cDNA-mRNA hybridization. A critical step in the procedure, which used biotinylated cDNA probes, was the selection of fixation conditions which maximized RNA retention and maintenance of tissue morphology. Tissues fixed in modified Carnoy's fixative (58% ethanol, 30% choloroform, 10% acetic acid, 2% formaldehyde) for 2-4 hr at -20 degrees C sectioned well and retained their cell morphology and cytoplasmic RNA. Other treatments important for the procedure included demineralization in 0.25 M HCl and removal of matrix by hyaluronidase digestion. In situ hybridization with type-specific collagen cDNA probes revealed that type II collagen mRNA was present in cells throughout the SL calvaria. More importantly, cells with type II collagen mRNA were also present in N calvaria which do not synthesize the protein. The overall abundance of type II-positive cells in N calvaria was not significantly different from that in SL calvaria, but their distribution throughout the bones differed. In general, the regional distribution of type II cells was inversely correlated with the extent of matrix mineralization. In the N calvaria, cells containing collagen type II mRNA were absent in the extensively mineralized superior zone, but were found in the temporal zone which showed limited mineralization. On the other hand, in the SL calvaria, which were substantially undermineralized overall, cells with type II mRNA were found throughout the tissue. Interestingly, the overall ratio of type I cells to type II cells was approximately 50% higher in N calvaria. These findings suggest that collagen type mRNA expression in the chick embryonic calvarium is correlated with, and perhaps dependent on, the extent of tissue matrix mineralization.     

Improved method for detection of cellular transcripts by in situ hybridization. Detection of virus-specific RNA in rous sarcoma virus-infected cells by in situ hybridization to cDNA

Optimal conditions for the detection of complex RNA sequences in individual cells by in situ hybridization have been determined by using in vitro cultured quail embryonic cells infected with Rous sarcoma virus and a single-stranded 3H-cDNA probe of high specific activity complementary to the RSV genome. It is shown that fixation of target tissue can be suitably achieved by using glutaraldehyde at low concentration, and subjecting cytological preparations to heat post-fixation treatment. Conditions for removing unhybridized radioactive probe molecules by means of S1 nuclease are reported.