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K E Bernstein

Publications and source records attributed to K E Bernstein.

89 records · Page 5Linked to original sources
PubMed1989

Evolutionary conservation of splice sites in sterile C mu transcripts and of immunoglobulin heavy chain (IgH) enhancer region sequences.

The immunoglobulin JHC mu intron was cloned from genomic DNA of a VHa3 rabbit and a 1257 bp sequence which contains conserved enhancer and splice sites was determined. From positions 315 to 1257, there is approximately 72 and 67% similarity to available sequences of man and mouse, respectively (counting gaps as single changes at single positions). In earlier studies of rabbit cDNAs encoding immunoglobulin heavy chains, we found a C mu-encoding cDNA clone (pB3) derived from splenic mRNA of a Trypanosome-hyperimmunized rabbit (VHa1) which lacked VH, DH or JH sequences and had an unknown sequence 5' of that encoding C mu. Comparison of this cDNA sequence with the present cloned genomic DNA sequence has now revealed that the start of cDNA pB3 corresponds to a position 80 base pairs 3' of the conserved octamer motif of the rabbit heavy chain enhancer. This mRNA was spliced to the acceptor site of C mu using a donor site which was 635 bp 3' of the enhancer octanucleotide. Our sequence of pB3 indicates that in rabbit as in mouse, a "nontron" (33 stop codons in three reading frames) can be formed utilizing a conserved splice site to produce a spliced transcript. The presence of evolutionarily conserved splice donor sites in the intron sequences of rabbit, mouse and man suggests a functional role during B cell ontogeny.

Animals↗
PubMed1988

Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus.

Indirect immunofluorescence shows that purified T7 RNA polymerase, when microinjected into monkey kidney (Vero) cells, localizes predominantly in the cytoplasm. To direct active T7 RNA polymerase to the nucleus, we first created unique restriction sites at two locations within the cloned gene for T7 RNA polymerase, T7 gene 1 and then inserted into these sites a 36-bp synthetic nucleotide sequence encoding the SV40 T antigen nuclear location signal. Insertion of the nuclear location signal between codons 10 and 11 of T7 RNA polymerase has only minimal effect on transcription activity in Escherichia coli, but its insertion four codons from the C terminus abolishes activity. Fusion proteins having only foreign codons ahead of codon 11 also have transcription activity in E. coli. Such fusion proteins can be expressed transiently from plasmids microinjected into monkey cells, using SV40 expression signals, and detected by immunofluorescence. A fusion protein containing a nuclear location signal localized predominantly in the nucleus whereas those which lack the signal localize predominantly in the cytoplasm. Ability to direct T7 RNA polymerase to the nucleus may be an advantage in attempting to make this enzyme useful for selective transcription in eukaryotic cells.

Amino Acid Sequence↗
PubMed1988

The isolation of angiotensin-converting enzyme cDNA.

Angiotensin-converting enzyme (ACE) is an Zn(II)-containing dipeptidyl carboxypeptidase that converts angiotensin I to the potent vasoconstrictor, angiotensin II. Using oligonucleotide probes based on the amino acid sequence of mouse kidney ACE, cDNA encoding this protein has been isolated. One cDNA, ACE.31, encodes the N-terminal 332 amino acids of mouse ACE, a portion of the protein containing a putative 34-amino acid leader sequence and the N terminus of the mature protein. Northern analyses with cloned ACE cDNA revealed that both mouse kidney and lung express two ACE mRNAs, one of 4900 and another of 4150 bases. Southern analysis suggests that cDNA ACE.31 is the product of a single gene, and thus these data add evidence to the hypothesis that the converting enzymes produced by epithelial and endothelial cells are identical.

Amino Acid Sequence↗
PubMed1988

Partial protein sequence of mouse and bovine kidney angiotensin converting enzyme.

Angiotensin converting enzyme (ACE) plays an important role in the regulation of renal blood pressure by the hydrolysis of the inactive precursor peptide angiotensin I to the potent vasopressor angiotensin II. Renal ACE is a surface membrane protein of both endothelium and tubular epithelium. Enzymatically active ACE was isolated from renal homogenates by chromatography using an affinity column constructed by linking an ACE inhibitor, lisinopril, to Affi-Gel 15. Analysis of eluates from this column showed that ACE activity was increased greater than 500-fold. SDS-polyacrylamide gel electrophoresis demonstrated a single band of molecular weight 144 kD (mouse) and 149 kD (bovine). N-terminal amino acid sequence analysis revealed: (formula; see text) Though bovine ACE has one additional N-terminal amino acid, these two partial sequences are highly homologous (16 of 20 positions are identical). Mouse ACE was digested with trypsin and the peptides were isolated by reverse phase HPLC. Analysis of the amino acid sequences showed that these tryptic peptides were unique to ACE. Thus, we were able to isolate ACE from bovine and mouse kidneys and show that they had substantial structural homology. They were also quite similar to that from rabbit lung.

Amino Acid Sequence↗
PubMed1985

Identification of genes for the constant region of rabbit T-cell receptor beta chains.

We describe cDNA clones from thymus mRNA of a young rabbit that have sequences highly homologous to the human and murine T-cell receptor beta-chain constant region (C beta). In rabbit, man, and mouse there is a conserved extra cysteine in the constant region that could lead to a free thiol group or alternative disulfide bond formation depending on the locations and total numbers of cysteines in assembled receptor molecules. A cDNA clone (CL ANA 11) with 571 bases 5' of the C beta coding sequence has an open reading frame starting at a methionine codon that encodes 141 amino acids in frame with the C beta sequence. The encoded sequence has no resemblance to known immunoglobulin or beta-chain variable regions or other known proteins. An oligonucleotide probe from the 5' end of the encoded protein hybridizes to an approximately equal to 2-kilobase genomic DNA fragment that contains C beta gene sequences and to an approximately equal to 8-kilobase mRNA species in the thymus mRNA preparation from which the clone was derived. Within the 5' coding sequence there is a stretch of 211 bases containing strings of alternating purines and pyrimidines that may form Z-DNA. The sequence of the last 55 base pairs adjacent to C beta resembles the corresponding segment of mouse cDNA clone 86T3 that contains sequence 5' of the mouse C beta 1 gene. Although the function of a potential protein encoded by the 5' end of CL ANA 11 is unknown, it could play a role in regulation of thymocyte growth and differentiation.

Amino Acid Sequence↗
PubMed1985

Germline VH genes in an a3 rabbit not typical of any one VHa allotype.

We have undertaken investigations in the rabbit of VH genes that may be responsible for the observations of VHa allotypes unexpected from an animal's pedigree (latent allotypes). A short cDNA probe was prepared and shown to be specific for VHa2 mRNA. Southern analyses with short and large probes were unrevealing but screening of a lambda phage library from a VHa3-expressing animal identified a number of unusual genes. These VH genes are remarkable in that they are far closer to one another in the genome (in one case 3085 bps) than VH genes reported in mouse or man, they are highly homologous over long stretches of sequence, and they encode proteins not typical of any one VHa allotype. Proteins similar to the sort encoded by these genomic V-regions may explain some of the observations of latent allotype. Our data suggest that the allelic behavior of VHa allotypes is not due to allelism of a regulatory mechanism that acts upon identical VH genes in rabbits of different VHa phenotypes.

Animals↗
PubMed1984

Sequence of a cDNA encoding Basilea kappa light chains (K2 isotype) suggests a possible relationship of protein structure to limited expression.

We present the complete sequence of a cDNA encoding rabbit immunoglobulin kappa light chains of the Basilea isotype (K2). Although all rabbits seem to possess a K2 constant region gene, expression of this gene in most rabbits is minimal if present at all. Even in Basilea rabbits the majority of expressed immunoglobulins are of lambda type. We find that the sequence of our Basilea cDNA constant region and the sequence of a "silent" K2 gene from b4 rabbits (bas-N4) are almost identical. The bas (K2) isotype lacks cysteine at position 171 in the constant region that is present in all K1 constant regions and usually forms an interdomain disulfide bond, with a cysteine at position 80 of the variable region. We postulate that one factor contributing to the low expression of the bas (K2) isotype could be a paucity of V kappa regions lacking cysteine at position 80. If a typical rabbit V kappa encoding Cys at position 80 is rearranged and expressed with th K2 isotype. B cells with mRNAs encoding light chains with free sulfhydryl groups would result. These cells may fail to form functional immunoglobulin receptors. Only a small subset of rabbit variable regions that lack the cysteine at position 80 would rearrange and encode K2 light chains lacking a free sulfhydryl group.

Amino Acid Sequence↗
PubMed1984

The structural and genetic basis for expression of normal and latent VHa allotypes of the rabbit.

The immunoglobulin heavy chain variable regions of the rabbit are unusual in having genetically controlled, serologically detectable alternative forms, the VHa allotypes, as well as minor VH allotypes of the x, y and w groups. New insights into the probable structural basis for the VHa allotypes have come from re-examination of earlier protein sequence data in the light of newly deduced protein sequences derived from sequencing cloned cDNAs and genomic DNAs encoding VH regions. Here we review this sequence information, and define the allotype-correlated differences at seven positions in framework region 1 and 10 positions in framework region 3 that may lead to the serologically detectable allotypic determinants (allotopes). Most alternative amino acids at allotype-correlated positions can be derived from each other by single-base changes. Thus somatic mutations and/or gene conversion-like events must be considered along with other serological and genetic explanations for various reported observations of the production of latent VHa allotypes. The proximity of rabbit VH genes (approximately 3 kb apart) might enhance the likelihood of conversion-like events in both germline and somatic cells.

Amino Acid Sequence↗
PubMed1984

Kappa-chain allotypes and isotypes in the rabbit: cDNA sequences of clones encoding b9 suggest an evolutionary pathway and possible role of the interdomain disulfide bond in quantitative allotype expression.

The constant regions of rabbit kappa light chains are unusual because the sequences of the allotypic forms can differ more from each other than do some variable regions with which they associate. We report the nucleic acid sequence of a full-length cDNA clone of b9 allotype and show comparisons to available sequences of the rabbit kappa allotypes b4, b5, and bas-N4. Our analyses suggest that the primordial rabbit kappa gene encoded a bas-like sequence. They also reveal a surprising difference in the position of the variable region cysteine that forms the interdomain disulfide bond that is unique to most rabbit kappa chains. One b9 cDNA sequence lacks the usual cysteine-80 and instead encodes cysteine-108, which in three-dimensional models appears capable of forming the interdomain disulfide bond with cysteine-171 in the constant region. A partial sequence of a second b9 clone encodes both cysteine-80 and cysteine-108; the translation product of this clone could have a free reactive sulfhydryl group that might lead to an unstable nonfunctional Ig molecule. The fact that pre-B cells with b9 kappa chains do not differentiate and expand into productive Ig-producing cells with frequencies comparable to the other allotypes may be explained if a substantial proportion of the gene products have a free sulfhydryl group. Our sequence results suggest that in cells differentiating to produce kappa light chains of b9 allotype the number and location of the cysteines influence immunoglobulin expression.

Animals↗
PubMed1984

Complete sequence of a cloned cDNA encoding rabbit secreted mu-chain of VHa2 allotype: comparisons with VHa1 and membrane mu sequences.

The complete sequence of a cDNA clone (pmu3) encoding a secreted IgM heavy chain from a rabbit of VHa2 allotype has been determined. Comparison of the nucleic acid and amino acid sequences with a comparable mouse mu-chain shows unusually high homologies of the sequences of the fourth domains and secretory portions of the molecules (80 to 90% vs 56 to 73% for the other domains). This high degree of homology is also seen with the sequences of the fourth domain and secreted terminus of human mu-chain. The DNA sequence of a second cDNA clone encoding the membrane form of rabbit IgM is approximately 90% homologous with the mouse and human coding sequences for the membrane terminus, and there is 98 and 97% amino acid sequence homology. We found an unusually long (156 base pair) 5' untranslated region in clone pmu3 and discovered that 105 bases at the 5' end were complementary to the terminal portion of the CH4 domain of our mu sequence. Southern blotting analyses and the finding of a 13 base pair segment in rabbit genomic V region DNA that is homologous to the complementary strand of a segment of the CH4 of rabbit mu strongly suggests that this extended sequence resulted from a reverse transcriptase "hooking error". We have also obtained and sequenced another cDNA clone from a VHa1 cDNA library. Comparison of the V region of clone pmu3 from VHa2 with the 117 available positions of a1 DNA sequence shows 25 differences, nine of which are in allotype-associated codons. There is also a two-codon deletion in the VHa1 compared to VHa2 in the third framework region that may contribute to allotypic differences. Development of VHa allotype-specific probes, now in progress, is complicated by the fact that most of the allotype-associated amino acids of a1, a2, and a3 can be derived from each other by single base changes.

Amino Acid Sequence↗
PubMed1983

The sequences of rabbit kappa light chains of b4 and b5 allotypes differ more in their constant regions than in their 3' untranslated regions.

We report the sequence of a cDNA clone encoding the entire variable and constant regions of a rabbit kappa light chain of b5 allotype. The deduced amino acid sequence of the variable region (positions 1-95) is 86% homologous to that of a b4 light chain protein [BS-1) (1) but the b4 and b5 constant regions are only 74% homologous. Comparison of this DNA sequence to that of a cDNA clone encoding a b4 constant region shows that the kappa allotypes b4 and b5 have diverged significantly more in their coding region than in the 3' untranslated regions (86% vs 96% nucleotide sequence homologies). This implies either a function for the 3' untranslated region with evolutionary pressures to conserve or an accelerated divergence of the coding regions.

Amino Acid Sequence↗
PubMed1983

Nucleotide sequence of a rabbit IgG heavy chain from the recombinant F-I haplotype.

We report the sequence of a cDNA encoding a rabbit immunoglobulin gamma heavy chain of d12 and e14 allotypes with high homology to partial cDNA sequences from rabbits of d11 and e15 allotypes. The encoded rabbit protein shows homologies with human (68-70%) and mouse (60-63%) gamma chains. The nucleotide sequence homologies of the CH domains range from 76-84% with human and 64-76% with mouse sequences. Comparison of the portion of VH encoding amino acid positions 34-112 with a previously determined VH sequence of the same allotype shows high conservation of sequences in the second and third framework segments but more marked differences both in length and encoded amino acids of the second and third complementarity-determining regions (CDRs). We also found a high degree of homology with a human genomic V-region, VH26 (77%) and a remarkable similarity between rabbit and human second CDR sequences and human genomic D minigenes. These results provide additional evidence that D minigene sequences share information with the CDR2 portion of VH regions.

Alleles↗
PubMed1983

Use of Trypanosoma equiperdum infected rabbits as a source of splenic mRNA; construction of cDNA clones and identification of a rabbit mu heavy chain clone.

Rabbits were infected by Trypanosoma equiperdum and the splenic mRNA was isolated. In vitro translation of this RNA and immunoprecipitation with anti-light chain, anti-heavy chain, anti-mu and anti-VH antibodies demonstrated that T. equiperdum infection elicits large quantities of splenic mRNA encoding mu and kappa chains. The mu and gamma heavy chains and the kappa light chains synthesized in the cell-free translation system were specifically immunoprecipitated by antisera to heavy chain VHa and light chain kappa b allotypes. In vitro labeling of spleen cells from trypanosome-infected animals demonstrated that the biosynthetically labeled IgM has a mu chain of higher molecular weight than the mu chain synthesized by in vitro translation, a difference that is largely abolished when cellular glycosylation is blocked with the antibiotic tunicamycin. Enrichment for heavy chain or light chain mRNA was achieved by fractionating mRNA from trypanosome-infected animals on a sucrose gradient. cDNA clones carrying mu heavy chain sequences were produced using a 'one tube' protocol and identified by cross species hybridization and hybridization selection. Infection of rabbits with T. equiperdum followed by sucrose gradient enrichment of splenic mRNA has provided sufficient quantities of mRNA encoding mu heavy chain suitable for cDNA cloning.

Animals↗
PubMed1982

Nodular (pseudosarcomatous) fasciitis, a nonrecurrent lesion: clinicopathologic study of 134 cases.

Clinicopathologic review of 134 patients originally diagnosed as having nodular (pseudosarcomatous) fasciitis is presented. In 114 patients with 116 lesions, no recurrence of the lesion was noted. Of the 114 patients, 85% were younger than 50 years of age, and the forearm and arm were the most common sites of presentation. Nonrecurrent lesions rarely exceed 4 cm and 71% were smaller than 2 cm. In at least six instances, incompletely resected lesions never recurred. Though all lesions were histologically reminiscent of reparative mesenchymal tissue, four subtypes--the reactive type, the densely cellular type, those with osteoid or cartilaginous metaplasia, and the so-called proliferative fasciitis--were distinguished from the majority of lesions that conform to the description given by Kornwaler. Recurrence of the tumor was noted in 18 patients. Fifteen of 18 lesions recurred within two years, and two more recurred at 30 months following initial excision. In all these cases, review of the histology and clinical course led to a revision of the original diagnosis. The greatest number of errors was made in incorrectly classifying of inflammatory fibrous histiocytoma. Recurrence of a lesion originally diagnosed as nodular fasciitis should lead to a careful reappraisal of the pathologic findings.

Adolescent↗