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E E Max

Publications and source records attributed to E E Max.

At least 55 records · Page 3Linked to original sources
PubMed1989

Negative control region at the 5' end of murine leukemia virus long terminal repeats.

Using in vitro protein binding and in vivo functional studies, we have identified novel regulatory sequences near the 5' end of murine leukemia virus (MuLV) long terminal repeats (LTRs). These sequences are highly conserved in all MuLV LTRs as well as in feline leukemia virus and gibbon ape leukemia virus LTRs. In this upstream conserved region (UCR), gel retardation assays detected two overlapping but distinct binding sites (UCR-U and UCR-L) for nuclear proteins (UCRF-U and UCRF-L). Three lines of evidence suggest a negative regulatory role for the UCR in viral transcription: (i) an inverse correlation was found between MuLV transcripts and nuclear proteins binding the UCR in the spleens of five different mouse strains; (ii) in vivo treatment of NFS mice with lipopolysaccharide resulted in the induction of splenic viral transcripts and the concomitant disappearance of UCR-binding proteins; and (iii) in mouse L cells transfected with an MuLV LTR linked to the chloramphenicol acetyltransferase (CAT) gene, cotransfected UCR oligonucleotides increased CAT expression, presumably by competing for inhibitory trans-acting factors.

Animals↗
PubMed1988

Identification and partial purification of a protein binding to the human immunoglobulin kappa enhancer kappa E2 site.

We previously described a domain in the 5' half of the human immunoglobulin kappa enhancer which could bind nuclear proteins in vitro, as detected by a lambda exonuclease protection assay. A second more 3' binding domain in the enhancer has now been detected by a similar assay employing a different exonuclease, the T7 gene 6 exonuclease. Using this assay and starting with a pig spleen nuclear extract, we have purified 5000-fold a protein that binds to the 3' domain. In a DNase I footprint experiment the partially purified protein protects a 27 bp segment in the enhancer centered around the sequence CAGGTGGC, which corresponds to the kappa E2 sequence motif described in the mouse kappa enhancer. The protein, designated NF-kappa E2, also appears to bind at a position downstream of kappa E2, at or near the kappa E3 site. Proteins capable of binding at kappa E2 are found in several mammalian species and are expressed in both lymphoid and non-lymphoid tissues.

Animals↗
PubMed1988

Activation of the human immunodeficiency virus long terminal repeat by herpes simplex virus type 1 is associated with induction of a nuclear factor that binds to the NF-kappa B/core enhancer sequence.

It has been previously shown that herpes simplex virus type 1 (HSV-1) infection of HeLa cells results in augmentation of gene expression directed by the human immunodeficiency virus (HIV) long terminal repeat (LTR). This effect is presumably mediated by protein interactions with the LTR. We have used two different assays of DNA-protein interactions to study the HSV-induced activation of the HIV LTR. Activation of the HIV LTR is associated with increased protein binding to LTR sequences in a region including the NF-kappa B/core enhancer and the Sp1 binding sequences as monitored by an exonuclease protection assay. Gel retardation assays demonstrated that HSV-1 infection resulted in the induction of a nuclear factor(s) that binds to the NF-kappa B/core enhancer sequence. In addition to the activation of the HIV LTR, HSV induction of NF-kappa B activity may be important for the regulation of HSV gene expression during a herpesvirus infection.

Base Sequence↗
PubMed1988

High-resolution analysis of the human gamma-globin gene promoter in K562 erythroleukemia cell chromatin.

We performed high-resolution mapping studies of the DNAse I-hypersensitive sites located just 5' to the human G gamma- and A gamma-globin genes of K562 erythroleukemia cells, in which these genes are constitutively expressed at low levels. This analysis revealed that the hypersensitive site extends from approximately -210 +/- 5 to -25 +/- 5 base pairs (bp) upstream from the transcription initiation site. Within this region, a GC-rich region located between the proximal CCAAT box and the TATA box is particularly accessible to nuclease digestion; however, the 5' end of the hypersensitive site is less accessible to nucleases. The pattern of DNAse I cleavage does not change on either strand with hemin induction of K562 cells, which increases the rate of gamma-globin gene transcription about threefold. The region within the hypersensitive site includes all the consensus promoter elements of the gamma-globin genes as well as an octamer sequence located between -182 and -175, and a region associated with a variety of mutations that may cause hereditary persistence of fetal hemoglobin (HPFH).

Chromatin↗
PubMed1987

Human immunoglobulin kappa gene enhancer: chromatin structure analysis at high resolution.

The murine immunoglobulin kappa gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific approximately 0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine kappa enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5' end of the nuclease-susceptible region.

B-Lymphocytes↗
PubMed1987

B-cell nuclear proteins binding in vitro to the human immunoglobulin kappa enhancer: localization by exonuclease protection.

Proteins capable of interacting with the enhancer of the immunoglobulin kappa gene in vitro have been detected in extracts of nuclei from human B cells and from human, mouse, and rabbit spleens. The experiments, based on an exonuclease protection technique, demonstrate nuclear protein factors binding to a 30- to 35-base-pair domain containing both the simian virus 40 enhancer core element (TTTCCA) and the octamer CAGGTGGC that was previously identified as the consensus sequence for protein-binding sites in the murine immunoglobulin heavy-chain enhancer. This 30- to 35-base-pair domain in the human kappa enhancer is homologous to a site of protein binding detected in the murine kappa enhancer by other investigators using a gel retardation assay. Our results complement in vivo dimethyl sulfate footprinting studies of the human immunoglobulin kappa enhancer which demonstrated B cell-specific changes in guanine reactivity immediately 5' to the consensus octamer. Together, these findings suggest that DNA-binding proteins in B-cell nuclei interact with the 5' portion of the human kappa-gene enhancer. Such proteins could play a role in the B cell-specific transcription of the human immunoglobulin kappa gene.

B-Lymphocytes↗
PubMed1986

The rabbit kappa 1 b5 immunoglobulin gene: another J region gene cluster with only one functional J gene segment?

Our previous analysis of an immunoglobulin gene encoding the rabbit kappa chain of b4 allotype revealed that of the five J-like sequences in the J kappa cluster of this gene, only one, J2, appeared to be functional. This unusual ratio of J pseudogenes to functional J genes is unique among all J clusters of light and heavy chain genes of all species examined to date, including the cluster from the rabbit kappa 2 isotype, and must have consequences for diversity generation of b4 immunoglobulins. The fact that the only two known b5 J kappa sequences are different from the functional J2 of the b4 allotype prompted investigation of the b5 J kappa cluster to determine whether it resembled the b4 cluster, or the more typical mouse or human J kappa clusters. Our analysis of the b5 gene reveals a J kappa cluster strikingly similar to that of b4; apparent defects occur in all J sequences except J2. Although J2 is apparently functional, it differs from the J2 of the b4 locus by four nucleotide and three amino acid substitutions. The unusually high degree of sequence similarity previously observed between the b4 and b5 loci in the noncoding (vs the coding) regions extends through the newly sequenced DNA segment and remains an enigma.

Animals↗
PubMed1986

Human J chain gene: chromosomal localization and associated restriction fragment length polymorphisms.

Gene clones encoding the human J chain, the protein that links immunoglobulin monomers, were recently described. Using probes from J chain clones we have now investigated the chromosomal location of this gene by analysis of somatic cell hybrids and by in situ chromosome hybridization. The gene is located on the long arm of chromosome 4 in band q21, the chromosomal band in which a consistent translocation with chromosome 11 has been observed in some acute leukemias. An additional human sequence that cross-hybridizes with some J chain gene probes is located on a different chromosome. Restriction fragment length polymorphisms deriving from length variations in a tandemly repeated region 5' to the J chain gene were detected; these should facilitate the analysis of genetic linkage between this gene and other markers on chromosome 4.

Chromosome Mapping↗
PubMed1985

Human J chain gene. Structure and expression in B lymphoid cells.

As part of an ongoing investigation of the regulation of gene expression in B cell development, we have obtained a genomic DNA clone encoding the human J chain protein. The nucleotide sequence of exons encoding the mature protein defines a 137 amino acid primary sequence similar to that previously determined at the protein level. Probes from the gene have been used to analyze J chain expression in human cell lines corresponding to pre-B and B lymphocytes. J chain RNA was detected in two of six human pre-B cell lines and in 8 of 10 B cell lines expressing various Ig isotypes. The expression of the J chain gene is, thus, not tightly linked to IgM or IgA secretion. Our data do not, however, support the recent suggestion (7) that synthesis of J chain precedes that of mu chain in B lymphocyte differentiation. Because of the presence of nine candidate polyadenylation signals (AATAAA or AATTAAA) downstream of the C-terminal coding block of the J chain gene, the 3' end of the gene could not be determined from sequence data alone. To define the 3' end, J chain RNA from a human B lymphocyte line was used to protect an end-labelled DNA fragment from S1 nuclease digestion. The sequence 40 basepairs 5' of the functional polyadenylation site identified by these S1 experiments is homologous the same region of a previously reported mouse J chain complementary DNA clone.

Amino Acid Sequence↗
PubMed1985

Structure of a functional rabbit class I MHC gene: similarity to human class I genes.

Studies of rabbit major histocompatibility complex proteins have suggested that rabbits express only a single class I antigen, in contrast to most mouse strains (H-2K, D, and L) and man (HLA-A, -B, and -C), which express three. To explore the significance and the molecular basis of this apparent species difference, we have characterized the expressed class I protein and a corresponding cDNA clone from the rabbit cell line RL-5, which is derived from the inbred B/J rabbit strain. As an extension of these analyses, this report documents the genomic sequence of a gene, designated 19-1, which encodes the same histocompatibility antigen expressed in RL-5. The availability of the corresponding full-length cDNA and amino-terminal protein sequence indicates the fully functional nature of the 19-1 gene and allows presumptive assignment of the transcription start site and delineation of exon/intron boundaries. Comparisons of the rabbit gene with homologous human and mouse sequences reveal a striking similarity between 19-1 and human genes in both exon/intron organization and specific nucleotide sequences; this close similarity allows tentative identification of previously unrecognized transcriptional start sites in the human genes.

Animals↗
PubMed1985

Rabbit-mouse hybridomas secreting intact rabbit immunoglobulin.

Rabbit-mouse hybridomas offer the potential for production of monoclonal rabbit antibodies by immortal cell lines. In previous studies, it was possible to produce and stabilize rabbit-mouse hybrid cells secreting either a rabbit heavy or light chain. These have been useful for structural characterization of the individual rabbit immunoglobulin polypeptides and for isolation of large amounts of immunoglobulin mRNA for molecular studies. For some studies, however, it would be useful to have intact rabbit immunoglobulin molecules comparable to the myeloma proteins available in the human and mouse. The availability of rapid, sensitive and specific assays for rabbit heavy and light chains and allotypes located on specific chains has now permitted the early identification of clones secreting intact rabbit immunoglobulin. Vigorous cloning efforts have resulted in isolation and partial stabilization of three such clones. The first, H105, secretes a product with a kappa light chain bearing the b6 allotype and a mu-chain bearing the a1 allotype. Biochemical and serologic analyses of the product show that it is secreted as a fully assembled IgM pentamer and that the rabbit heavy and light chains are covalently associated. No rabbit J-chain gene was detected in H105 by Southern blot analysis. The second hydridoma, H134, secretes a product with a mol. wt of 150 K, consisting of a b4 light chain and an a1 heavy chain. The third, H171, secretes an alb4 IgG with antibody specificity for group C streptococcal carbohydrate. An additional rabbit-mouse hybridoma, H89, have been produced which secretes a rabbit heavy chain lacking group a allotypic activity. The rabbit heavy chain, which is associated with a mouse light chain, has an N-terminal amino acid sequence identical to a2-positive molecules although thorough serologic analysis revealed no group a allotypic activity.

Amino Acid Sequence↗
PubMed1984

CG dinucleotide clusters in MHC genes and in 5' demethylated genes.

In the DNA of higher vertebrates the dinucleotide CG is unique in two respects: it occurs far less frequently than would be expected on the basis of the content of cytosine and guanine in a given DNA segment ("CG suppression") and it contains predominantly 5-methyl-cytosine, the only modified nucleotide common in vertebrate DNA. Here we point out the existence of CG clusters, i.e. localized lapses in the usual CG suppression, in two categories of DNA segments from vertebrates: around the polymorphic exons of major histocompatibility complex (MHC) genes and in the 5' regions of certain other genes. These observations contradict the recent suggestion that CG frequency is uniform over long contiguous segments of DNA containing several genes. A model for the origin of these CG clusters as a consequence of regional demethylation of germline DNA is supported by analysis of other sequence features of these regions as well as by previously published data on the methylation status in sperm DNA of two of these CG-rich regions.

Animals↗
PubMed1984

A genomic gene encoding the b5 rabbit immunoglobulin kappa constant region: implications for latent allotype phenomenon.

We previously reported that domestic rabbits of five immunoglobulin kappa allotype strains (b4v, b4, b5, b6, and b9) harbor at least two DNA sequences that hybridize strongly to kappa constant region probes in Southern blots. One of these sequences ("type A") has been identified as encoding the constant region of the kappa 2 isotype, an immunoglobulin chain that most rabbits express only at low levels, if at all. We identified the second sequence--for rabbits of the b4 allotype--as encoding the nominal b4 kappa chain (or kappa 1 isotype), but for rabbits of other allotypes no definite identification for this "type B" sequence could be made. Here we suggest that the type B sequence in rabbits of the other domestic allotypes also encodes the nominal kappa 1 immunoglobulin chain. We show this directly for the b5 allotype; a type B sequence cloned from b5 DNA has been found to contain an apparently functional gene encoding the b5 constant region sequence. Indirect arguments suggest the corresponding conclusion for the b4v, b6, and b9 allotypes. We have considered the implications of these results for the phenomenon of "latent allotype" expression.

Animals↗
PubMed1984

Rabbit class I MHC genes: cDNA clones define full-length transcripts of an expressed gene and a putative pseudogene.

The rabbit cell line RL-5 was recently found to express only a single detectable major histocompatibility complex class I protein, in contrast to the multiple class I proteins expressed in cells of the mouse (K, D, and L) and human (A, B, and C). To clarify this difference in the rabbit major histocompatibility complex, we have begun an investigation of the class I genes expressed in the RL-5 cell line and here report the construction of 31 class I cDNA clones derived from RL-5 mRNA. Restriction endonuclease mapping has allowed classification of these clones into five groups, apparently representing five distinct class I mRNA transcripts. The sequence of a full-length cDNA from the predominant group (representing 26 of the 31 clones) reveals a typical class I structure, with an amino terminus that exactly matches the 36 amino terminal residues previously determined for the class I protein immunoprecipitated from RL-5. Complete sequence of a second distinct class I mRNA transcript was deduced from cDNA clones of the second largest group (representing two of the 31 clones). Although this transcript corresponds to a complete class I sequence, an anomaly in initiation sequence may preclude its translation in the proper reading frame. Comparison of the rabbit class I sequences to those of mouse and human revealed significantly higher homologies between the rabbit and the human than between mouse and rabbit or mouse and human sequences.

Amino Acid Sequence↗
PubMed1984

Structure of a germline rabbit immunoglobulin V kappa-region gene: implications for rabbit V kappa-J kappa recombination.

Rabbit kappa-immunoglobulin chains exhibit diversity in the number of amino acids between the invariant residues Cys 88 and Phe 98; this length diversity is formally similar to that found in the human and mouse heavy chain systems, in which it results from interposition of the D element between V and J. To explore the molecular basis for this length diversity in rabbit kappa-chains we have determined the nucleotide sequence of a rabbit germline V kappa immunoglobulin gene. The spacing between the 7-mer and 9-mer signal elements of this gene suggest that it could recombine with J kappa without a D element. We discuss alternative explanations for the length diversity of rabbit kappa-chains.

Amino Acid Sequence↗
PubMed1983

Structural analysis of a rabbit immunoglobulin kappa 2 J-C locus reveals multiple deletions.

We previously reported that domestic rabbits harbor at least two DNA sequences that hybridize strongly to immunoglobulin kappa C region probes in Southern blots. One of these was cloned from a domestic b4 rabbit and identified as the gene for the nominal b4 allotype kappa chain which is expressed at high levels. We now have cloned (from a b4 rabbit) the other homologous sequence and find that it encodes a kappa chain nearly identical to the kappa 2 chain of "bas" rabbits, which is not normally expressed at detectable levels in domestic rabbits. Sequence analysis of this kappa 2 chain reveals a J kappa -C kappa locus with no obvious coding sequence defects that could explain its low expression. However, several base changes in a putative enhancer region as well as deletions (totalling about 1.5 kb) in the J-C intron might be related to low expression. The comparison between these two kappa genes raises questions about the selective pressures operating during the evolution of this gene system.

Amino Acid Sequence↗
PubMed1983

cDNA clone encoding a complete rabbit immunoglobulin kappa light chain of b4 allotype.

We have cloned a cDNA encoding a kappa light chain of the b4 allotype. The clone, pB4D5, was derived from the allotype-specific mRNA of a rabbit-mouse hybridoma 12F2, which secretes a monoclonal rabbit b4 kappa chain. Nucleotide sequence analysis of the entire pB4D5 insert reveals complete variable (V) and constant (C) regions and portions of the signal peptide and 3' untranslated region. The translated pB4D5 region amino acid sequence matches the NH2-terminal sequence of the kappa chain secreted by 12F2 and is similar, though not identical, to several other b4 kappa sequences. A V region-specific probe isolated from pB4D5 hybridizes to more than 25 bands on a genomic Southern blot of b4 rabbit DNA and yields a similar pattern with DNA from other allotypes. Sequence comparisons between pB4D5, a cloned germ-line precursor of the pB4D5 joining (J) region, and published b4 amino acid sequences reveal a peculiar feature of rabbit kappa V-J recombination in which this system resembles mouse and human heavy chain recombination more than any known kappa system.

Amino Acid Sequence↗