Differential staining of human and mouse chromosomes in interspecific cell hybrids

Bobrow, Martin; Cross, Jane

doi:10.1038/251077a0

Cited by 180 publications

(69 citation statements)

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“…Human chromosomes are preferentially lost in fusions with CHO or mouse cells, and the use of adequate selective conditions allows the retention of specific human genes (14,23,28,30,31). Precise identification of chromosomes or portions of chromosomes is possible with the use of banding and differential staining techniques (3,28,31,39) and isozyme marker analysis (25,32 (23,30,31,38).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

Lobatón¹,

Moreno²,

Oxender³

1984

Mol. Cell. Biol.

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We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, tsO25C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, tsO25C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20. Amino acid accumulation in mammalian cells is carried out by several distinct transport systems. The major components for neutral amino acid uptake in mammalian cells are systems A, ASC, and L, which were first identified in Ehrlich ascites tumor cells (5, 26). System A is Na+ dependent and serves mainly for the uptake of amino acids with short, polar, or linear side chains. System L is Na+ independent, shows reactivity toward branched-chain and aromatic amino acids, and exhibits rapid exchange between intracellular and extracellular substrates. System ASC is Na+ dependent and shows a strong preference for alanine, serine, and cysteine. Similar systems have been characterized in a number of different cell lines (1,4,9,13,16,27). Recently, transport systems A, ASC, and L have been characterized in Chinese hamster ovary (CHO) cells (2,34). Studies on system L regulation has also been carried out in these cells (24,33,(35)(36)(37) by using a temperature-sensitive leucyl-tRNA synthetase mutant, the CHO tsHl line. When this mutant is incubated at marginally permissive temperatures, a significant increase in leucine transport activity occurs, indicating that the cell responds to leucine limitation resulting from the defective leucyl-tRNA synthetase. Elevated leucine levels in the growth medium, however, allow CHO tsHl cells to grow at nonpermissive temperatures, indicating that increased uptake of leucine is associated with temperature resistance in this mutant. In ad...

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Section: Discussionmentioning

confidence: 99%

Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

Lobatón¹,

Moreno²,

Oxender³

1984

Mol. Cell. Biol.

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“…The remaining hybrids were derived from Chinese hamster cells (21). Our independent verification of the human chromosomes in the hybrid cell lines was done by Giemsa-11 staining, which differentially stains rodent and human chromosomes (6,37), and in some cases also by fluorescence in situ hybridization. At least 15 metaphases were analyzed for each hybrid cell line with the exception of GM11010 and GM10478, in which cases 12 and 11, respectively, were analyzed.…”

Section: Materuils and Methodsmentioning

confidence: 99%

A human Alu RNA-binding protein whose expression is associated with accumulation of small cytoplasmic Alu RNA.

Chang¹,

Nelson²,

Bilyeu³

et al. 1994

Mol. Cell. Biol.

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Human Alu sequences are short interspersed DNA elements which have been greatly amplified by retrotransposition. Although initially derived from the 7SL RNA component of signal recognition particle (SRP), the Alu sequence has evolved into a dominant transposon while retaining a specific secondary structure found in 7SL RNA. We previously characterized a set of Alu sequences which are expressed as small cytoplasmic RNAs and isolated a protein that binds to these transcripts. Here we report that biochemical purification of this protein revealed it as the human homolog of the SRP 14 polypeptide which binds the Alu-homologous region of 7SL RNA. The human cDNA predicts an alanine-rich C-terminal tail translated from a trinucleotide repeat not found in the rodent homolog, which accounts for why the human protein-RNA complex migrates more slowly than its rodent counterpart in RNA mobility shift assays. The human Alu RNA-binding protein (RBP) is expressed after transfection of this cDNA into mouse cells. Expression of human RBP in rodent x human somatic cell hybrids is associated with substantial increase in endogenous small cytoplasmic Alu and scBl transcripts but not other small RNAs. These studies provide evidence that this RBP associates with Alu transcripts in vivo and affects their metabolism and suggests a role for Alu transcripts in translation in an SRP-like manner. Analysis of hybrid lines indicated that the Ahu RBP gene maps to human chromosome 15q22, which was confirmed by Southern blotting. The possibility that the primate-specific structure of this protein may have contributed to Alu evolution is considered.Alu sequences are short transposed elements endogenous to the human genome (24, 55). These elements have accumulated to nearly 1 million copies comprising approximately 5% of human DNA. Alu repeats have sequence homology with and are believed to have been derived from the Alu-homologous region of the 7SL RNA component of signal recognition particle (SRP) (64,69,70,71). The current model of Alu evolution suggests that two independently derived Alu-homologous domains of 7SL dimerized to form the more efficient transposing element known as Alu (26,28,45,46,47). Since then, Alu repeats have continued to evolve within primate genomes. A major advance in understanding Alu mobility was the result of work by researchers in several laboratories who classified Alu repeats into subfamilies according to their evolutionary relatedness as determined by sequence homology (8,27,45,59,72). This, together with recent phylogenetic evidence, indicates that multiple Alu source genes continue to spawn new transposed elements (22, 25, 31, 39, 42, 67; reviewed in reference 49).Why genetic elements with disruptive potential have been allowed to proliferate to high copy number in human DNA remains enigmatic. Perhaps Alu mobility improves adaptability of the host genome, or perhaps Alu DNA elements or their RNA products have adopted a function(s) which more directly benefits their host cells (9,19,51,74). Given their short lengt...

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“…All human/ rodent somatic cell hybrids grown for DNA preparations (Table 1), have been recharacterized as to human chromosome composition by karyotyping (39,40), human isozyme analysis (41,42), or both. High molecular weight DNA from human, rodent and hybrid clones was digested with the restriction enzymes EcoRI, Pst I (Boehringer Mannheim), or Taq I (P-L Biochemicals).…”

Section: Methodsmentioning

confidence: 99%

Nucleotide sequence of cDNA encoding human alpha 2-macroglobulin and assignment of the chromosomal locus.

Kan¹,

Solomon²,

Belt³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Six a2-macroglobulin (a2M) cDNA clones were isolated from a human liver cDNA library by using synthetic oligonucleotides as hybridization probes. One of these, pa2M1, carries a 4.6-kilobase-pair insert, which was sequenced. The insert contains the coding sequences for the mature a2M polypeptide (1451 amino acids) and for a 23-amino acid signal peptide at the NH2 terminus of the precursor proa2M. At the 3' end of the insert a poly(A) addition signal A-A-T-A-A-A and part of the poly(A) tail of the messenger RNA were found. The protein sequence deduced from the nucleotide sequence agrees with the published a2M amino acid sequence for all except three residues. The a2M locus was assigned to human chromosome 12 by Southern blot analysis with DNA from a panel of mouse/human somatic cell hybrids, using a2M cDNA as a hybridization probe.a2-Macroglobulin (a2M) is a serum glycoprotein and a major plasma proteinase inhibitor with a wide specificity. a2M-related proteins are present in all vertebrate species (1-4). Human a2M is a tetramer of four identical 185-kDa subunits, arranged as a pair of dimers each consisting of two disulfidelinked monomers (5, 6). The a2M polypeptide has a so-called bait region and an internal thiol ester bond, which account for its properties as a proteinase inhibitor. The bait region, composed of a series of target peptide bonds for plasma proteinases (7, 8) (13,14).This suggests that the conformational change, which accompanies the complex formation between a2M and proteinases and the hydrolysis of the thiol ester bond, exposes regions of the a2M molecule that are recognized by these receptors. Receptor-mediated endocytosis of proteinase-a2M complexes by macrophages and liver cells leads to clearance of the complexes from the circulation.Internal thiol ester bonds are also found in the complement proteins C3 and C4 (15), which are derived from precursor polypeptides of size similar to a2M (180-200 kDa). Their thiol ester sites are found in positions comparable to those in a2M, and the amino acid sequences of all three thiol ester sites are conserved. These observations led to the proposal that the C3, C4, and a2M genes are derived from a common ancestral gene (16). The sequences of murine and human C3 and human C4 and partial cDNA sequences of murine C4 have recently been determined (17-21). Comparison of human a2M with murine C3 revealed a 25% overall sequence homology (17,18,22

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Differential staining of human and mouse chromosomes in interspecific cell hybrids

Cited by 180 publications

References 5 publications

Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

A human Alu RNA-binding protein whose expression is associated with accumulation of small cytoplasmic Alu RNA.

Nucleotide sequence of cDNA encoding human alpha 2-macroglobulin and assignment of the chromosomal locus.

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