离体培养下大豆体细胞胚胎发生的组织学研究

大豆胚状体可以直接从未成熟的子叶表皮及表皮下面1—3层细胞发生。这些细胞经过脱分化后,首先形成细胞质浓厚、核大的胚的发生细胞,胚发生细胞再分裂形成胚性细胞团,胚性细胞团再继续分裂形成胚状体。胚状体的发育过程和合子胚一样,经过球形、心形,鱼雷期和子叶期等诸阶段发育成小植株。此外,在诱导胚状体发生过程中,还观察到另一值得注意的现象:在未成熟胚的子叶表皮下面1至较深处的数层细胞,也转变成分生状细胞团,这些分生状细胞团呈不规则状,从其起源看,可称它们为内生“胚状体”,这些内生“胚状体”培养至20天,即停止生长发育。     

Synteny mapping in the bovine: genes from human chromosome 5.

In an effort to generate a more complete bovine syntenic map of Type I comparative anchor loci, seven homologs to genes found on HSA5 were mapped using a panel of bovine x rodent hybrid somatic cells. Five HSA5 genes, CSF2, RPS14, PDGFRB, FGFA, and CSF1R, were assigned to bovine syntenic group U22 (chromosome 7), while two others, C9 and HGMCR, mapped to U10 and U5, respectively. Previous studies had assigned the HSA5 marker SPARC to bovine syntenic group U22. The mapping of genes spanning the length of HSA5 in cattle and also in mouse permits syntenic comparisons between prototypic genomes of three mammalian orders, providing insight into the evolutionary history of this region of the ancestral mammalian genome.     

Synteny between the loci for a novel FACIT-like collagen locus (D6S228E) and alpha 1 (IX) collagen (COL9A1) on 6q12-q14 in humans.

A 1.8-kb cDNA encoding portion of a novel collagenous chain was isolated from a human rhabdomyosarcoma cell line by cross-hybridization using a chicken type V collagen probe. Sequence analysis suggests that this chain belongs to the recently discovered group of collagens, termed the FACIT class of macromolecules. This cDNA was used to locate the corresponding gene (D6S228E) to chromosome 6, notably at position 6q12-q14. Interestingly, within this region of human chromosome 6 residues the alpha 1 (IX) collagen gene (COL9A1), a member of the FACIT group.     

Chromosomal assignment of 46 brain cDNAs.

Expressed sequence tags (ESTs) have been obtained from several hundred brain cDNAs as an initial effort to characterize expressed brain genes. These ESTs will become tools for human genome mapping and they will also provide candidate causative genes for inherited disorders affecting the central nervous system. We have developed a procedure for the rapid chromosomal assignment of these ESTs: cDNA sequences are first analyzed by a computer program to determine regions likely not to be interrupted by introns in the genomic DNA. A pair of oligonucleotide primers is then designed to amplify this region by the polymerase chain reaction using DNA template from human-rodent somatic cell hybrid chromosomal panels. The chromosomal assignment of the cDNA is determined by studying the segregation of the amplified products in these panels. In this paper we describe the mapping of 46 brain ESTs, as well as observations on the amplification of rodent sequences.     

Somatic cell hybrids, sequence-tagged sites, simple repeat polymorphisms, and yeast artificial chromosomes for physical and genetic mapping of proximal 17p.

Somatic cell hybrids retaining the deleted chromosome 17 from 15 unrelated Smith-Magenis syndrome (SMS) [del(17)(p11.2p11.2)] patients were obtained by fusion of patient lymphoblasts with thymidine kinase-deficient rodent cell lines. Seventeen sequence-tagged sites (STSs) were developed from anonymous markers and cloned genes mapping to the short arm of chromosome 17. The STSs were used to determine the deletion status of these loci in these and four previously described human chromosome 17-retaining hybrids. Ten STSs were used to identify 28 yeast artificial chromosomes (YACs) from the St. Louis human genomic YAC library. Four of the 17 STSs identified simple repeat polymorphisms. The order and location of deletion breakpoints were confirmed and refined, and the regional assignment of several probes and cloned genes were determined. The cytogenetic band locations and relative order of six markers on 17p were established by fluorescence in situ hybridization mapping to metaphase chromosomes. The latter data confirmed and supplemented the somatic cell hybrid results. Most of the hybrids derived from [del(17)(p11.2p11.2)] patients demonstrated a similar pattern of deletion for the marker loci and were deleted for D17S446, D17S258, D17S29, D17S71, and D17S445. However, one of them demonstrated a unique pattern of deletion. This patient is deleted for several markers known to recognize a large DNA duplication associated with Charcot-Marie-Tooth (CMT) disease type 1A. These data suggest that the proximal junction of the CMT1A duplication is close to the distal breakpoint in [del(17)(p-11.2p11.2)] patients.     

The L-isoaspartyl/D-aspartyl protein methyltransferase gene (PCMT1) maps to human chromosome 6q22.3-6q24 and the syntenic region of mouse chromosome 10.

We have mapped the genes for the human and mouse L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77) using cDNA probes. We determined that the human gene is present in chromosome 6 by Southern blot analysis of DNA from a panel of mouse-human somatic cell hybrids. In situ hybridization studies allowed us to confirm this identification and further localize the human gene (PCMT1) to the 6q22.3-6q24 region. By analyzing the presence of an EcoRI polymorphism in DNA from backcrosses of C57BL/6J and Mus spretus strains of mice, we localized the mouse gene (Pcmt-1) to chromosome 10, at a position 8.2 +/- 3.5 cM proximal to the Myb locus. This region of the mouse chromosome is homologous to the human 6q24 region.     

Differentiation of myoblasts and CNS cells grown either separately or as co-cultures on microcarriers

Dispersed neuronal and muscular elements from fetal or neonatal origin, can organize and mature in culture when grown on positively charged cylindrical microcarriers (MCS), to a stage which simulatein vivo maturation. Cells arrange themselves on the MCS to form aggregates which remain floating in the nutrient medium. In such a tridimensional organization, the neuronal tissue is capable of regenerating a network of nerve fibers which establish synapse interconnections and undergo myelination. Oligodendrocytes organize on MCS in a tridimensional pattern and produce extensive myelin-like membranes. Myoblasts in MC-cultures fuse into polynucleated myotubes which become striated and contract spontaneously. Creatine kinase and acetylcholine receptor (AChR) are formed during myogenesis in similar quantities in MC-cultures and in monolayers. When both neuronal and muscle tissues are prepared from the same fetus (autologous nerve-muscle co-cultures) and are cultured on MCS, they interconnect to form neuro-muscular junctions. Cells from both tissues, exhibit better differentiation, for longer periods in MC-cultures than they do in monolayers. The floating functional entities are easy to sample and can be harvested for ultrastructural, immunocytochemical and biochemical analysis. In addition, MC-cultures can be used as a good tool for the study of acute and chronic exposures to toxicological agents, as well as for implantation into demyelinated, injured or dystrophic tissues. In this case the MCS in the implanted entities will serve as identifiable markers.