Gene for human insulin receptor: localization to site on chromosome 19 involved in pre-B-cell leukemia.

Consistent chromosomal translocations in neoplastic cells may alter the expression of proto-oncogenes that are located near the breakpoints. The complementary DNA sequence of the human insulin receptor is similar to those of the EGF receptor (erbB oncogene) and products of the src family of oncogenes. With in situ hybridization and Southern blot analysis of somatic cell hybrid DNA, the human insulin receptor gene was mapped to the distal short arm of chromosome 19 (bands p13.2----p13.3), a site involved in a nonrandom translocation in pre-B-cell acute leukemia.

Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways.

A new family of protein kinase C-related genes has been identified in bovine, human, and rat genomes. The alpha-, beta-, and gamma-type protein kinase sequences are highly homologous, include a kinase domain, and potential calcium-binding sites, and they contain interspersed variable regions. The corresponding genes are located on distinct human chromosomes; the possibility of even greater genetic complexity of this gene family is suggested by Northern and Southern hybridization analyses.

Cloning, sequencing, and expression of the gene coding for the human platelet alpha 2-adrenergic receptor.

The gene for the human platelet alpha 2-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the purified receptor. The identity of this gene has been confirmed by the binding of alpha 2-adrenergic ligands to the cloned receptor expressed in Xenopus laevis oocytes. The deduced amino acid sequence is most similar to the recently cloned human beta 2- and beta 1-adrenergic receptors; however, similarities to the muscarinic cholinergic receptors are also evident. Two related genes have been identified by low stringency Southern blot analysis. These genes may represent additional alpha 2-adrenergic receptor subtypes.

The neu gene: an erbB-homologous gene distinct from and unlinked to the gene encoding the EGF receptor.

The neu oncogene, identified in ethylnitrosourea-induced rat neuroglioblastomas, had strong homology with the erbB gene that encodes the epidermal growth factor receptor. This homology was limited to the region of erbB encoding the tyrosine kinase domain. It was concluded that the neu gene is a distinct novel gene, as it is not coamplified with sequences encoding the EGF receptor in the genome of the A431 tumor line and it maps to human chromosome 17.

Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene.

A novel potential cell surface receptor of the tyrosine kinase gene family has been identified and characterized by molecular cloning. Its primary sequence is very similar to that of the human epidermal growth factor receptor and the v-erbB oncogene product; the chromosomal location of the gene for this protein is coincident with the neu oncogene, which suggests that the two genes may be identical.

Characterization of a novel 350-kilodalton nuclear phosphoprotein that is specifically involved in mitotic-phase progression.

A gene assigned to human chromosome 1q32-41 encodes a novel protein of 3,113 amino acids containing an internal tandem repeat of 177 amino acids. The protein, which we have named "mitosin," was identified by direct binding to purified retinoblastoma protein in vitro with a region distantly related to the retinoblastoma protein-binding site of E2F-1. Mitosin is expressed throughout S, G2, and M phases of the cell cycle but is absent in G1. Its localization is dramatically reorganized from a rather homogeneous nuclear distribution in S phase to paired dots at the kinetochore/centromere region, to the spindle apparatus, and then to the midbody during M-phase progression. This spatial reorganization coincides closely with the temporal phosphorylation patterns of mitosin. Overexpression of N-terminally truncated mutants blocks cell cycle progression mainly at G2/M. These results suggest that mitosin may play an important role in mitotic-phase progression.

Comparison of nonerythroid alpha-spectrin genes reveals strict homology among diverse species.

The spectrins are a family of widely distributed filamentous proteins. In association with actin, spectrins form a supporting and organizing scaffold for cell membranes. Using antibodies specific for human brain alpha-spectrin (alpha-fodrin), we have cloned a rat brain alpha-spectrin cDNA from an expression library. Several closely related human clones were also isolated by hybridization. Comparison of sequences of these and other overlapping nonerythroid and erythroid alpha-spectrin genes demonstrated that the nonerythroid genes are strictly conserved across species, while the mammalian erythroid genes have diverged rapidly. Peptide sequences deduced from these cDNAs revealed that the nonerythroid alpha-spectrin chain, like the erythroid spectrin, is composed of multiple 106-amino-acid repeating units, with the characteristic invariant tryptophan as well as other charged and hydrophobic residues in conserved locations. However, the carboxy-terminal sequence varies markedly from this internal repeat pattern and may represent a specialized functional site. The nonerythroid alpha-spectrin gene was mapped to human chromosome 9, in contrast to the erythroid alpha-spectrin gene, which has previously been assigned to a locus on chromosome 1.

Allele loss at the retinoblastoma locus in human ovarian cancer.

To gain a broad spectrum on allelic loss of specific loci in ovarian tumors, we initially examined DNA from 23 pairs of ovarian tumors and matched peripheral blood lymphocyte samples from the same patients, using 27 polymorphic DNA markers distributed on 13 chromosomes. Significant high frequency of allelic deletion (22%-44%) at chromosome 13 loci (D13S31, D13S32, D13S33, and D13S34) at bands q12-q34 was observed in tumor tissues. These results led us to investigate the loss of heterozygosity at the retinoblastoma (RB) locus in ovarian tumors, because the RB gene is a tumor-suppressor gene located at 13q14. Analysis of the variable number of tandem repeat sequence polymorphism in intron 20 in the RB gene revealed that 6 (30%) of 20 patients with informative samples showed allelic loss at the RB locus in their tumor tissues. This loss, of relatively high frequency, suggests that the RB gene, or a closely linked gene, seems to be involved in the development of ovarian cancer.

Genetic analysis of benign, low-grade, and high-grade ovarian tumors.

Genetic abnormalities were assessed in 56 benign, low-, and high-grade ovarian tumors using comparative genomic hybridization (CGH) and analysis of loss of heterozygosity (LOH). In addition, 95 epithelial tumors were analyzed for microsatellite repeat instability. DNA sequence copy number abnormalities (CNAs) were not detected in the benign tumors, and more were detected in high-grade than in low-grade cancers. Almost no microsatellite repeat instability was detected in these cancers. CNAs occurring in more than 30% of the cancers included increased copy number on 3q25-26 and 8q24 and reduced copy number on 16q and 17pter-q21. Another 14 CNAs occurred in more than 20% of the cancers. Increased copy number at 3q25-26 and 20q13 was the most frequent CNA in low-grade tumors, and increased copy number at 8q24 occurred preferentially in high-grade tumors. The presence of a large number of CNAs per tumor was significantly correlated with reduced patient survival duration. Reduced copy number on 17pter-q21 was most strongly associated with accumulation of a large number of CNAs. The overall concordance between LOH and reduced copy number detected by CGH was 84%, but only 31% of the LOH was associated with reduced copy number detected using CGH.

Proton/peptide cotransporter (PEPT 2) from human kidney: functional characterization and chromosomal localization.

We report here on the functional characterization of the H+/peptide cotransporter PEPT 2 cloned from human kidney and on the chromosomal localization of the PEPT 2 gene. PEPT 2, when functionally expressed in HeLa cells, induces the transport of the neutral dipeptide glycylsarcosine. The induced transport activity is markedly influenced by extracellular pH. The optimum pH for the transport process is 6.0-7.0. Kinetic analysis has revealed that PEPT 2 is a high-affinity transporter, the Michaelis-Menten constant for glycylsarcosine being 74 +/- 14 microM. The human intestinal H+/peptide cotransporter PEPT 1 has 4-fold less affinity for the dipeptide under identical experimental conditions. Studies with other chemically diverse dipeptides have established that PEPT 2 possesses higher affinity than PEPT 1 not only for neutral peptides but also for peptides consisting of anionic and/or cationic amino acids. Somatic cell hybrid analysis and in situ hybridization have shown that the gene encoding PEPT 2 maps to human chromosome 3q13.3-q21.

Human skeletal muscle insulin receptor substrate-1. Characterization of the cDNA, gene, and chromosomal localization.

Insulin receptor substrate-1 is a major substrate of insulin receptor Tyr kinase. We have now cloned the IRS-1 cDNA from human skeletal muscle, one of the most important target tissues of insulin action, localized and cloned the human IRS-1 gene, and studied the expression of the protein in Chinese hamster ovary cells. Human IRS-1 cDNA encodes a 1242 amino acid sequence that is 88% identical with rat liver IRS-1. The 14 potential Tyr phosphorylation sites include 6 Tyr-Met-X-Met motifs and 3 Tyr-X-X-Met motifs that are completely conserved in human IRS-1. Human IRS-1 has > 50 possible Ser/Thr phosphorylation sites and one potential ATP-binding site close to the NH2-terminal. The human IRS-1 gene contains the entire 5'-untranslated region and protein coding region in a single exon and was localized on chromosome 2 q36-37 by in situ hybridization. By Northern blot analysis, IRS-1 mRNA is rare and consists of two species of 6.9 and 6 kilobase. By using quantitative polymerase chain reaction after reverse transcription of total RNA from human fetal tissues, IRS-1 mRNA could be identified in all tissues. When human IRS-1 cDNA was expressed in Chinese hamster ovary cells, the protein migrated between 170,000-180,000 M(r) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was rapidly Tyr phosphorylated upon insulin stimulation. Thus, IRS-1 is widely expressed and highly conserved across species and tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression profile and chromosomal location of cDNA clones, identified from an enriched adult retina library.

PURPOSE: To delineate the profile of genes expressed in the adult human retina and assign chromosomal location of cDNA clones. METHODS: The end-sequence of random clones from an enriched human retinal cDNA library was analyzed by NCBI database search. Expression profile was established by northern blot analysis, database search, or both. Selected cDNA clones were localized to human chromosomes by somatic cell hybrid analysis, in situ hybridization to metaphase chromosomes, or both. Chromosomal location was also obtained by searching the databases. RESULT: One hundred and thirty-seven clones were isolated from the subtracted retinal library. Fifty-one clones were identical with 35 known human genes in GenBank, and 24 clones corresponded to 23 uncharacterized human expressed sequenced tags (ESTs), novel genes, or both. The remaining 59 clones were not pursued further because they contained bacterial sequences or repetitive elements. Several clones indicated a restricted pattern of expression with high levels of transcripts in the retina. Chromosomal location of novel retinal ESTs is also reported. CONCLUSIONS: This study provides a profile of genes expressed in the adult human retina. One round of subtraction eliminated most constitutively expressed genes and permitted partial normalization of the retinal library. Twenty-three novel genes were identified. The combined information obtained from expression analysis and chromosomal localization of retinal cDNAs should be valuable in identifying candidate genes for diseases involving retinal dysfunction.

A complex chromosome rearrangement with at least five breakpoints studied by fluorescence in situ hybridization.

A newborn infant with multiple congenital anomalies was diagnosed with an unbalanced translocation of chromosomes 1 and 5. Studies of parental chromosomes revealed a complex rearrangement in the patient's mother involving the exchange of terminal long arms between chromosomes 1 and 5 and the insertion of an interstitial segment from the same chromosome 5q into chromosome 2q by high-resolution G-banding. Further study of the mother's chromosomes by fluorescent in situ hybridization (FISH) detected an additional insertion between the rearranged chromosomes 2 and 5, which was not revealed by G-banding. This led to the identification of a complex translocation-insertion between 3 chromosomes with at least 5 breaks [t(1;5;2)(1pter--> 1q42.3::5q23.2-->5qter;5pter-->5q21.2:: 2q33--> 2q35::1q42.3-->1qter;2pter-->2q33::5q21 .2--> 5q23.2::2q35-->2qter)] and illustrates the value of FISH as an adjunct to standard cytogenetics, particularly in cases of complex rearrangements.

Antenatal diagnosis of 45,X/48,XYYY.

A diagnosis of 45,X/48,XYYY was made antenatally. Counseling this case was hampered by the paucity of literature describing the phenotype of patients with this chromosome constitution. The fetus had ambiguous external genitalia, a horseshoe kidney, a cerebral cortical cyst and arachnodactyly.

Mosaic dup (9p) diagnosed by fluorescence in situ hybridization (FISH).

We describe a girl with some manifestations of the dup (9p) syndrome. High-resolution Giemsa-banded karyotype of her lymphocytes documented that she was mosaic with 80% of cells being 46,XX, and 20% 46,XX,-20, + der(20;?) (p13;?). The additional material on 20p could not be defined clearly by high-resolution Giemsa banding, as the banding pattern appeared consistent with either distal 9p or distal 13q. In order to make a definitive cytogenetic diagnosis, we used fluorescence in situ hybridization (FISH) with a chromosome 9 specific DNA library to establish that the origin of the additional chromosomal material on chromosome 20 was from 9p. FISH used in this situation enabled us to counsel the family specifically regarding the prognosis and manifestations of distal 9p duplication.

"Pseudomosaicism" for 4p- in amniotic fluid cell culture proven to be true mosaicism after birth.

Pseudomosaicism is noted in approximately 1% of amniotic fluid cell studies. Some represent numerical abnormalities, but pseudomosaicism for structural chromosomal abnormalities is also seen. Pseudomosaicism is not usually considered clinically significant. Recently, we evaluated a 13-month-old girl with developmental delay and minor anomalies suggestive of 4p- syndrome. In 5 of 100 peripheral lymphocytes, she had a deletion 46,XX,del(4)(p15). Review of a prenatal amniocentesis study performed on the mother of our patient disclosed that one colony of 18 examined from 2 in situ cultures had the same abnormality, whereas none of the 27 cells from a flask culture showed the abnormality. Results of this study had originally been reported as showing pseudomosaicism. To our knowledge, amniotic fluid pseudomosaicism of a structural abnormality has not previously been shown to reflect true mosaicism in fetal tissue or liveborn children. The actual incidence of this phenomenon is unknown, but it may be present in unexamined children with minimal clinical findings. Apparently only one previous case of mosaic 4p- in a liveborn individual has been reported.

FISH diagnosis of partial trisomy 13 and tetrasomy 13 in a patient with severe trigonocephaly (C) phenotype.

An infant girl with manifestations resembling Optiz trigonocephaly (C) syndrome who died at age 6 days was found to have a complex chromosome abnormality with t(13;18)(q22;q23) and a recombinant chromosome 13 involving duplicated segments of 13q. Precise characterization was possible with the application of fluorescence in situ hybridization (FISH) using chromosome specific probes. The patient's phenotype is compared to that of other syndromes involving trigonocephaly.

Novel isodicentric chromosome 18 in an abnormal infant with a mosaic karyotype [46,XY/46,XY,-18,+dic(18)(q12.2]).

A previously unreported isodicentric chromosome 18 was discovered in an abnormal infant boy whose mosaic karyotype was 46,XY/46,XY,-18,+idic(18)(q12.2). His constellation of congenital anomalies was typical of the 18q-syndrome. The clinical and cytogenetic characteristics of this patient are reported, and the literature concerning isochromosomes of 18 is reviewed.

Familial dup(5)(q15q21) associated with normal and abnormal phenotypes.

We studied a familial dup(5q) present in a phenotypically normal father and his monozygotic twin daughters with different abnormal phenotypes. High-resolution chromosome analysis suggested that the duplicated segment was of region q15-21, which seems to be the smallest dup(5q) reported thus far. This dup(5q) was confirmed by fluorescence in situ hybridization with a chromosome 5 painting library and 5q cosmid clones. The presence of the dup(5q) in a normal father suggested that the duplication itself may be harmless. The anomalies in the twins may be due to processes other than this chromosome change.

Duplication of euchromatin without phenotypic effects: a variant of chromosome 16.

A family with an unusual variant of chromosome 16 is presented. The mother and son both with additional material present in the short arm of chromosome 16 adjacent to the centromere are phenotypically normal. The extra C-band negative region has been shown not to be composed of alpha satellite DNA. The literature regarding other familial cases of what appears to be the same variant of chromosome 16 is reviewed.